julián esteban muñoz henao

JULIÁN ESTEBAN MUÑOZ HENAO

Avaliação do Peptídeo Sintético (P10): associado ao

tratamento quimioterápico em camundongos BALB/c

anérgicos infectados com Paracoccidioides brasiliensis

Tese apresentada ao Programa de Pós-Graduação em Microbiologia do Instituto de Ciências Biomédicas da Universidade de São Paulo, para obtenção do Título de Doutor em Ciências.

São Paulo 2012

JULIÁN ESTEBAN MUÑOZ HENAO

Avaliação do Peptídeo Sintético (P10): associado ao

tratamento quimioterápico em camundongos BALB/c

anérgicos infectados com Paracoccidioides brasiliensis

São Paulo 2012

Tese apresentada ao Programa de Pós-Graduação em Microbiologia do Instituto de Ciências Biomédicas da Universidade de São Paulo, para obtenção do Título de Doutor em Ciências. Área de concentração: Microbiologia Orientador: Prof. Dr. Carlos Pelleschi Taborda

Versão original

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Serviço de Biblioteca e Informação Biomédica do

Instituto de Ciências Biomédicas da Universidade de São Paulo

© reprodução total

Muñoz-Henao, Julián Esteban. Avaliação do peptídeo sintético (P10) associado ao tratamento quimioterápico em camundongos BALB/c anérgicos infectados com Paracoccidioides brasiliensis / Julián Esteban Muñoz-Henao. -- São Paulo, 2012. Orientador: Prof. Dr. Carlos Pelleschi Taborda. Tese (Doutorado) – Universidade de São Paulo. Instituto de Ciências Biomédicas. Departamento de Microbiologia. Área de concentração: Microbiologia. Linha de pesquisa: Estudo da Imunidade humoral e celular nas infecções causadas por fungos sistêmicos. Versão do título para o inglês: Evaluation of synthetic peptide (P10) associated with chemotherapy treatment in immunosuppressed mice BALB/c infected with Paracoccidioides brasiliensis. 1. P10 2. Paracoccidioidomicose 3. P. brasiliensis 4. Adjuvante I. Taborda, Prof. Dr. Carlos Pelleschi II. Universidade de São Paulo. Instituto de Ciências Biomédicas. Programa de Pós-Graduação em Microbiologia III. Título.

ICB/SBIB0196/2012

UNIVERSIDADE DE SÃO PAULO INSTITUTO DE CIÊNCIAS BIOMÉDICAS

______________________________________________________________________________________________________________

Candidato(a): Julián Esteban Muñoz-Henao.

Título da Tese: Avaliação do peptídeo sintético (P10) associado ao tratamento quimioterápico em camundongos BALB/c anérgicos infectados com Paracoccidioides brasiliensis.

Orientador(a): Prof. Dr. Carlos Pelleschi Taborda.

A Comissão Julgadora dos trabalhos de Defesa da Tese de Doutorado, em sessão

pública realizada a ................./................./................., considerou

( ) Aprovado(a) ( ) Reprovado(a)

Examinador(a): Assinatura: ...............................................................................................

Nome: ....................................................................................................... Instituição: ................................................................................................

Examinador(a): Assinatura: ................................................................................................ Nome: .......................................................................................................

Instituição: ................................................................................................

Examinador(a): Assinatura: ................................................................................................

Nome: ....................................................................................................... Instituição: ................................................................................................

Examinador(a): Assinatura: ................................................................................................ Nome: .......................................................................................................

Instituição: ................................................................................................

Presidente: Assinatura: ................................................................................................

Nome: .......................................................................................................

Instituição: ................................................................................................

Aos meus pais e familiares, que me apoiaram no dia a dia e sempre

estiveram aí nos momentos difíceis da minha vida.

Meus pais Leslie Henao Torres e Fernando Muñoz Montoya que apesar da

distância, seguem sendo os melhores pais do mundo, me apoiando em todo

momento e responsáveis da minha formação e vida acadêmica. Meus irmãos, Pablo

Andrés e Margarita Maria, porque sempre me deram muito apoio e estimulo para

vencer. A minha esposa Nathalia Mejía Sánchez, que sempre esteve ao meu lado

em todos os momentos bons e difíceis, me dando todo carinho, amor, apoio e força

para continuar estudando e vivendo.

Todos são muito especiais para mim, são a força de cada dia na minha vida, graças por

existir...

AGRADECIMENTOS

Ao Professor Dr. Carlos Pelleschi Taborda, que foi e é exemplo de dedicação

e de excelência em pesquisa, agradeço de coração a oportunidade de trabalhar no

seu laboratório, obrigado pelos ensinamentos e o incentivo a cada dia. Obrigado por

ter me orientado e ensinado tantas coisas, Muito Obrigado Mesmo.

Ao Professor Gabriel Padilla, agradeço de coração a imensa ajuda para eu

fazer o estagio e minha pós-graduação aqui no Brasil.

Ao Professor Dr. Luiz R. Travassos, agradeço por todas as sugestões e

participações neste trabalho.

Aos Professores (as), Benedito Corrêa, Luis Carlos Ferreira, Maria Sueli S.

Felipe, Elaine G. Rodrigues, Gil Benard, Sandro Rogério de Almeida, Daniel de

Assis Santos, Bluma Faintuch, Sirlei Daffre pelas IMPORTANTES colaborações e a

todos os professores do Departamento de Microbiologia da Universidade de São

Paulo.

A todos os colegas e amigos do laboratório, Márcia Pinto da Silva, Felipe

Augusto, Oriana M. Nader, Diego Rossi, Leandro Buffoni e Martha Urán, Glauce M.

G. Rittner, Adriana Menezes, Fernanda Dias, Renata Amélia, Thor A. Sessa, Viviana

Sarria, Luciana Thomaz, Adelaide Galvão, Adriana Magalhães, Juliana de Amorim,

Paula Barbarian, Vinicius D. Luft, e Shirlei A. Vieira Marques.

Aos colegas da pós-graduação, Luis, Guilherme, Rafael, Aline, Fernando,

Catarina, Carolina, Lívia, Tati, galego, Inarei e Esther pelo convívio e apoio durante

esses anos de Pós-Graduação.

Ao técnico do biotério Carlos Augusto da Silva pelo cuidado dos animais

durante todos esses anos de pesquisa.

Aos secretários da Pós-Graduação e do Departamento de Microbiologia,

Elizabete Ribeiro, Alice Shimabuku, Bruno e Celso Pereira, Ana Maria Amaral e

Naíde Farripas, por sua amizade e gentileza no atendimento.

Aos funcionários da Sala de Esterilização: Elza, José, pela colaboração e

apoio técnico.

Aos funcionários do Serviço de Biblioteca do ICB pela ajuda, agilidade,

eficácia e gentileza no atendimento, especialmente a Renata Santos, Edilson,

Jacinta e Delza, que sempre estiveram ai quando eu pedia intensamente artigos,

que no conseguia pegar da internet. Obrigado por ser a melhor Biblioteca de todas!!

E finalmente, a todos que contribuíram direta e indiretamente para a

realização deste trabalho.

À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) pelo

apoio financeiro.

Obrigado por tudo!

E se estiver se arrastando na direção do cume de uma

montanha nunca escalada e vir outro grupo de

alpinistas subindo por um caminho paralelo? Quando se

faz ciência, pode-se sugerir um trabalho conjunto – a

cooperação parece tão mais criativa do que a

concorrência.

Maurice Wilkins, The Third Man of the Double Helix.

Na longa história da humanidade (e também dos

animais), aqueles que aprenderam a colaborar e a

improvisar de forma mais eficaz, foram os que

prevaleceram.

Charles Darwin

RESUMO

MUÑOZ, J. E. Avaliação do Peptídeo Sintético (P10): associado ao tratamento quimioterápico em camundongos BALB/c anérgicos infectados com Paracoccidioides brasiliensis. 2012. 231 f. Tese (Doutorado em Microbiologia) – Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2012. A paracoccidioidomicose (PCM), doença sistêmica de caráter granulomatoso, é causada pelo fungo termodimórfico Paracoccidioides brasiliensis. A PCM é endêmica na América Latina em paises como Argentina, Colômbia, Venezuela e Brasil e afeta principalmente pessoas das zonas rurais. Esta doença apresenta duas manifestações clinicas (aguda e crônica). Na maioria dos casos a PCM envolve primeiramente os pulmôes, podendo se disseminar para outros orgãos e sistemas. Os pacientes com PCM são submetidos ao tratamento medicamentoso com Sulfametoxazol/trimetoprim, Anfotericina B, e derivados azólicos. Por ser uma doença com altos índices de recidivas, longos períodos de tratamento são necessários. A glicoproteína de 43kDa (gp43), possui 416 aminoácidos, onde um trecho específico de 15 aminoácidos (QTLIAIHTLAIRYAN) designado como (P10), é reconhecido por linfócitos T. No presente trabalho, avaliamos a ativação da resposta imune e o efeito aditivo da imunização com o peptídeo P10, em camundongos induzidos à imunossupressão com dexametasona. Reações de HTT e contagem de leucocitos foram realizadas periodicamente para verificar o estado do sistema imune dos animais. Os resultados indicam um efeito aditivo da imunização com P10 e o tratamento com as drogas em camundongos BALB/c imunossuprimidos e infectados; associado a redução significativa da carga fúngica no pulmão, baço e fígado desses animais, detectamos aumento de citocinas proinflamatorias como IFN-γ, TNF-α e IL-

12, no homogenato de pulmão e no sobrenadante de cultura celular. Ensaios in vitro mostraram que a imunização com o P10 induziu uma efetiva resposta celular, com um aumento significativo na proliferação de linfócitos TCD4+ específicos provenientes de baço. Animais imunossuprimidos e imunizados com P10 apresentaram um aumento significativo na produção de Óxido Nítrico (NO) e foi observado a través de imunohistoquimica um aumento de células L3T4+, Ly-6G/Ly-6C+ e CD11b+ nos pulmões dos animais imunizados. A eficiencia na resposta levou a um aumento na sobrevida de 100%. Estes resultados sugerem que o peptídeo P10, pode atuar como adjuvante ao tratamento medicamentoso, representando uma alternativa promissória na geração de uma vacina anti-PCM e evitando possíveis recidivas no hospedeiro imunossuprimido. Palavras-chave: P10. Imunossupressão. P. brasiliensis. Paracoccidioidomicose.

Adjuvantes. Antifúngicos.

ABSTRACT

MUÑOZ, J. E. Assessment of Synthetic peptide (P10): associated with

chemotherapy treatment in BALB/c mice infected with Paracoccidioides brasiliensis. 2012. 231 p. Ph. D. thesis (Microbiology) – Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2012.

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease endemic of Latin America. It is caused by Paracoccidioides brasiliensis (Pb), a thermal dimorphic fungus. PCM is widespread mainly in Argentina, Colombia, Venezuela and Brazil affecting mainly rural workers. Inhalation of conidia is the provable route of infection. This sickness has multiple manifestations, and two progressive forms (acute and chronic) are recognized. In most cases, PCM involves primarily lungs and then disseminates to other organs and systems. PCM treatment is mandatory currently based on sulfametoxazole/trimethoprim, amphotericin B and azolic derivatives. Because it is a disease with high recurrence rate, long periods of treatment are necessary. The gp43 has 416-mer and mice and human T lymphocytes are stimulated by a 15-mer peptide designated as P10 (QTLIAIHTLAIRYAN). In the present work we evaluated the activation of immune responses and additive effect of P10 immunization in BALB/c mice induzed for immunossupression with Dexamethasone. HTT reactions and leucogram were performed periodically to check the status of the immune system of animals. The results indicate an additive effect of P10 immunization and treatment with drugs in BALB/c mice immunosuppressed and infected; associated with a significant reduction in fungal burden in the lung, spleen and liver of these animals, detected increase of proinflammatory cytokines such as IFN-γ, TNF-α e IL-12, in lung homogenates and cell culture supernatant. In vitro

assays have shown that P10 peptide immunization led to an effective cellular immune response increasing the lymphoproliferation of CD4+ specific lymphocytes from the spleen. Immunosuppressed animals that were immunized with P10 showed increased in the NO production by macrophages and were observed through the immunohistochemistry augmented of cells as L3T4+, Ly-6G/Ly-6C+ and CD11b+ in the lungs of immunized mice. The efficiency of response led to a 100% survival in animals immunized with peptide and treated with antifungal drugs. The present work shows that P10 can act as an adjuvant to drug treatment, represents a promising alternative for the generation of anti-PCM vaccines and avoiding possible recurrences in host immunocompromised. Keywords: P10. Immunosuppression. P. brasiliensis. Paracoccidioidomycosis. Adjuvant. Antifungal drugs.

LISTA DE ILUSTRAÇÕES

Figura 1 - Contagem do número de leucócitos no sangue periférico......................55

Figura 2 - HTT realizado em camundongos BALB/c...............................................56

Figura 3 - Sobrevida de camundongos BALB/c infectados i.t com 3x105 células leveduriformes de P. brasiliensis e tratados com dexametasona (0.15 mg/kg) na água de beber por 30 dias (●) grupo controle (○)....................................................57

Figura 4 - Baços de camundongos BALB/c submetidos ao tratamento com Dexametasona.........................................................................................................58 Figura 5 - UFCs de pulmão (P), baço (B) e fígado (F) de camundongos BALB/C

anérgicos, com 45 dias de infecção.........................................................................60

Figura 6 - UFCs de pulmão (P), baço (B) e fígado (F) de camundongos BALB/C anérgicos, com 60 dias de infecção.........................................................................61

Figura 7 - Dosagem da citocina IFN-γ, pelo método Cytometric Bead Array (CBA)

(FACSCalibur, EUA), de homogeneizado dos pulmões de camundongos BALB/c anérgicos, infectados por via intratraqueal com 3x105 células de P. brasiliensis e tratados com as drogas antifúngicas, associadas, ou não, à imunização com P10, e sacrificados com 45 e 60 dias de infecção...............................................................63 Figura 8 - Dosagem da citocina IL-12, pelo método Cytometric Bead Array (CBA)

(FACSCalibur, EUA), de homogeneizado dos pulmões de camundongos BALB/c anérgicos, infectados por via intratraqueal com 3x105 células de P. brasiliensis e tratados com as drogas antifúngicas, associadas, ou não, à imunização com P10, e sacrificados com 45 e 60 dias de infecção...............................................................64 Figure 9 - Dosagem da citocina TNF-α, pelo método Cytometric Bead Array (CBA)

(FACSCalibur, EUA), de homogeneizado dos pulmões de camundongos BALB/c anérgicos, infectados por via intratraqueal com 3x105 células de P. brasiliensis e tratados com as drogas antifúngicas, associadas, ou não, à imunização com P10, e sacrificados com 45 e 60 dias de infecção. .............................................................65 Figura 10 - Dosagem da citocina IL-10, pelo método Cytometric Bead Array (CBA)


(FACSCalibur, EUA), de homogeneizado dos pulmões de camundongos BALB/c anérgicos, infectados por via intratraqueal com 3x105 células de P. brasiliensis e tratados com as drogas antifúngicas, associadas, ou não, à imunização com P10, e sacrificados com 45 e 60 dias de infecção...............................................................67

Figura 12 - Dosagem da citocina IL-8, pelo método Cytometric Bead Array (CBA)

(FACSCalibur, EUA), de homogeneizado dos pulmões de camundongos BALB/c anérgicos, infectados por via intratraqueal com 3x105 células de P. brasiliensis e tratados com as drogas antifúngicas, associadas, ou não, à imunização com P10, e sacrificados com 45 e 60 dias de infecção...............................................................68 Figura 13 - Dosagem da quimiocina MCP-1, pelo método Cytometric Bead Array

(CBA) (FACSCalibur, EUA), de homogeneizado dos pulmões de camundongos BALB/c anérgicos, infectados por via intratraqueal com 3x105 células de P. brasiliensis e tratados com as drogas antifúngicas, associadas, ou não, à imunização com P10, e sacrificados com 45 e 60 dias de infecção........................69 Figura 14 – Dosagem da citocina IL-4, pelo método Cytometric Bead Array (CBA)


(FACSCalibur, EUA), de homogeneizado dos pulmões de camundongos BALB/c anérgicos, infectados por via intratraqueal com 3x105 células de P. brasiliensis e tratados com as drogas antifúngicas, associadas, ou não, à imunização com P10, e sacrificados com 45 e 60 dias de infecção...............................................................71 Figura 16 - Relação do perfil Th1/Th2, pelo método Cytometric Bead Array (CBA)

(FACSCalibur, EUA), de homogeneizado dos pulmões de camundongos BALB/c anérgicos..................................................................................................................72 Figura 17 - Detecção por ELISA, do isotipo IgG total presente no soro de

camundongos BALB/c anérgicos.............................................................................73 Figura 18 - Detecção por ELISA, do isotipo IgG2a total presente no soro de camundongos BALB/c anérgicos.............................................................................74 Figura 19 - Detecção por ELISA, do isotipo IgG2b total presente no soro de

camundongos BALB/c anérgicos.............................................................................75 Figura 20 - Detecção por ELISA, do isotipo IgG1 total presente no soro de camundongos BALB/c anérgicos.............................................................................76 Figura 21 - Determinação da produção de óxido nítrico no homogenato de pulmão

de camundongos BALB/c anérgicos imunizados ou não com o P10 e tratados ou não com as drogas antifúngicas...............................................................................77 Figura 22 - Determinação da produção de óxido nítrico no sobrenadante de cultura

celular de esplenocitos de camundongos BALB/c anérgicos imunizados ou não com o P10 e tratados ou não com as drogas antifúngicas..............................................78

Figura 23 - Identificação de células CD11b+ através de munohistoquímica dos

pulmões dos camundongos BALB/c anérgicos infectados com Pb18 e imunizados ou não com o P10....................................................................................................79 Figura 24 - Identificação de células Ly-6G/Ly-6C+ através de imunohistoquímica

dos pulmões dos camundongos BALB/c anérgicos infectados com Pb18 e imunizados ou não com o P10.................................................................................80 Figura 25 – Identificação de células L3T4+ através de imunohistoquímica dos

pulmões dos camundongos BALB/c anérgicos infectados com Pb18 e imunizados ou não com o P10...........................................................................................................81

Figura 26 - Quantificação de células do tecido pulmonar de camundongos BALB/c anérgicos infectados com Pb18 e imunizados ou não com o P10, marcadas pelo método de imunohistoquímica com os anticorpos Ly-6G/Ly-6C e L3T4..................82 Figura 27 - Ensaio de proliferação celular utilizando linfócitos esplênicos de animais imunizados com o P10 ou não imunizados 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis e animais controle (sham)....83 Figura 28 - Dosagem de IFN-γ, pelo método de ELISA em sobrenadante de cultura

de linfócitos esplênicos provenientes de animais imunizados com o P10, 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis....................84 Figura 29 - Dosagem de IL-12, pelo método de ELISA em sobrenadante de cultura de linfócitos esplênicos provenientes de animais imunizados com o P10, 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis....................85 Figura 30 - Dosagem de IL-10, pelo método de ELISA em sobrenadante de cultura de linfócitos esplênicos provenientes de animais imunizados com o P10, 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis....................86 Figura 31 - Dosagem de IL-4, pelo método de ELISA em sobrenadante de cultura de linfócitos esplênicos provenientes de animais imunizados com o P10, 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis.....................87 Figura 32 - Dosagem de IL-18, pelo método de ELISA em sobrenadante de cultura de linfócitos esplênicos provenientes de animais imunizados com o P10, 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis.....................88 Figura 33 - Dosagem de IL-1β, pelo método de ELISA em sobrenadante de cultura de linfócitos esplênicos provenientes de animais imunizados com o P10, 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis.....................89 Figura 34 - Dosagem de IL-17A, pelo método de ELISA em sobrenadante de cultura de linfócitos esplênicos provenientes de animais imunizados com o P10, 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis.............90 Figura 35 - Percentual de linfócitos esplênicos com fenótipo de memória.............91

Figura 36 - Percentual de linfócitos esplênicos com fenótipo de memória em

camundongos BALB/c anérgicos.............................................................................92 Figura 37 - Percentual de linfócitos esplênicos com fenótipo de reguladora em camundongos BALB/ c anérgicos............................................................................93 Figura 38 - Percentual de linfócitos esplênicos com fenótipo de reguladora em

camundongos BALB/c anérgicos. ...........................................................................94 Figura 39 – Contagem global de leucócitos do sangue periférico de camundongos BALB/c anérgicos.....................................................................................................95 Figura 40 - Curva de sobrevivência de camundongos BALB/c anérgicos infectados,

por via intratraqueal, com 3x105 leveduras de P. brasiliensis isolado 18 e imunossuprimidos, com dexametasona...................................................................96 Figura 41 - Curva de sobrevivência de camundongos C57/BL6 Knockout iNOS-/-

infectados por via intratraqueal com 3x105 leveduras de Pb18 e imunossuprimidos, com dexametasona..................................................................................................97 Figura 42 - Histopatologia dos pulmões dos camundongos BALB/c anérgicos

infectados com Pb18 e imunizados, ou não, com P10............................................98 Figura 43 – Detecção da fibrose pulmonar através da histopatologia dos pulmões de camundongos BALB/c imunossuprimidos e infectados com Pb18, imunizados, ou não, com P10....................................................................................................100 Figura 44 - Comparação da formação de fibras de colágeno do tipo I.................101 Figura 45 - Comparação da formação de fibras de colágeno do tipo III...............102 Figura 46 – Representação esquemática do desenho de um radiofármaco ........102 Figura 47 – Biodistribuição do 99mTc-HYNIC-P10 em camundongos BALB/c sadios.....................................................................................................................103 Figura 48 – Imagens cintilográficas em camundongos BALB/c 99mTc-HYNIC

ligado no peptídeo P10. ........................................................................................104 Figura 49 - Citotoxicidade in vivo do peptídeo P10. .............................................106 Figura 50 - Citotoxicidade in vitro do peptídeo P10...............................................107

LISTA DE ABREVIATURAS E SIGLAS

Abs Absorvância

Ac Anticorpo

AcM Anticorpo Monoclonal

B Baço

BHI Brain Heart Infusion (infusão de cérebro e coração)

BSA Soro Albumina Bovina

ºC Graus celcius

CD cluster of differentiation

CO2 Dióxido de carbono

DCs Células Dendríticas

Dex. Fosfato de Dexametasona

DMSO dimetilsulfóxido

D.O Densidade Ótica

DTH Testes cutâneos de hipersensibilidade tardia

E2 Estradiol

ELISA “Enzyme Linked Immuno Sorbent Assay”

EUA Estados Unidos da América

F Fígado

FcγR Receptor da fração Fc das imunoglobulinas

g Gramas

g Relative centrifuge force-Gravities

gp Glicoproteina

gp43 Glicoproteína de 43 kDa

HUVEC Human Umbilical Vein Endothelial Cells

HYNIC 6-hidrazinonicotinamida

H2O Água

HTT Hipersensibilidade Tipo Tardio

H2O2 Peróxido de hidrogênio

H2SO4 Ácido Sulfúrico

HCl Ácido clorídrico

HLA Antígeno Leucocitário Humano

HPLC High Performance Liquid Chromatography

IFN Inteferon gama

Ig Imunoglobulina

IgA Imunoglobulina A

IgG1 Imunoglobulina G isótipo 1

IgG3 Imunoglobulina G isótipo 3

IgG2a Imunoglobulina G isótipo 2a

IgG2b Imunoglobulina G isótipo 2b

IL-4 Interleucina 4

IL- 5 Interleucina 5

IL-10 Interleucina 10

IL-12 Interleucina 12

iNOS Óxido Nítrico Sintase

i.t. Intratraqueal

KDa Quilodalton

Kg Kilogramas

K2HPO4 Fosfato de potássio

KO “Knockout”

L-DOPA L-3,4 dihidroxifenilalanina

M Molar

MEFs Mouse Embryonic Fibroblasts

mg Mili gramos.

mM Mili Molar

mg Miligramas

mL Mililitros

min Minutos

MMcM Meio líquido quimicamente definido

MS Mass Spectrometry

MS Ministério de Saúde

MTT Thiazolyl Blue Tetrazolium Bromide

Mø Macrófagos

μl Microlitros

μg Microgramas

μm Micrometros

N Normal

NaCl Cloreto de sódio

NF-KB Nuclear Factor-KappaB

NK natural killer

nm nanômetro

NO Óxido Nítrico

P10 Peptídeo 10

Pb Pacoccidioides brasiliensis

Pb 18 Pacoccidioides brasiliensis isolado 18

PBS Fosfate buffer saline

PBMC Células mononucleares de sangue periférico

PCM Paracoccidioidomicose

pH Potencial Hidrogeniônico

PMNs polimorfonucleares

PRRs Receptores de reconhecimento padrão

rpm Rotações por minuto

SDS-PAGE Sodium Dodecyl Sulfate de PolyAcrylamide Gel

SPF Specific Pathogen Free

SZM-TMP sulfametoxazol-trimetoprim

99mTc Tecnécio-99 metaestável

TCD4+ Linfócito T CD4 positivo

TGF-β Transforming Growth Factor-beta

Th1 Linfócito T “helper” 1

Th2 Linfócito T “helper” 2

TNF-α Fator de necrose tumoral-alfa

Treg Células T Reguladoras

UFC Unidade formadora de colônia

VIH Vírus da Imunodeficiência Humana

SUMÁRIO

1 INTRODUÇÃO .......................................................................................................23

1.1 Paracoccidioidomicose.....................................................................................23

1.2 Agente etiológico...............................................................................................27

1.3 Fatores de virulência..........................................................................................29

1.4 Paracoccidioidomicose e o Sistema imune.....................................................32

1.5 Dexametasona e sistema imune.......................................................................35

1.6 Tratamento na Paracoccidioidomicose............................................................36

1.6.1 Derivados azólicos............................................................................................36

1.6.2 Derivados sulfonamídicos.................................................................................37

1.6.3 Polienos.............................................................................................................37

2 JUSTIFICATIVA......................................................................................................39

3 OBJETIVOS ...........................................................................................................40

3.1 Objetivo geral......................................................................................................40

3.2 Objetivos específicos.........................................................................................40

4 MATERIAL E MÉTODOS ......................................................................................41

4.1 Animais................................................................................................................41

4.2 Imunossupressão dos camundongos BALB/c................................................41

4.3 Avaliação do estado de imunossupressão......................................................41

4.3.1 Leucograma.......................................................................................................41

4.3.2 Reação de HTT.................................................................................................42

4.4 Isolado e Cultivo do Paracoccidioides brasiliensis........................................42

4.5 Infecção e Terapia..............................................................................................42

4.5.1 Preparo do inóculo............................................................................................42

4.5.2 Determinação da viabilidade das leveduras......................................................42

4.5.3 Infecção intratraqueal (i.t)………………………………………..………………....43

4.5.4 Terapia com agentes antimicrobianos...............................................................43

4.5.4.1 Primeiro protocolo..........................................................................................43

4.5.4.2 Segundo protocolo.........................................................................................44

4.6 Síntese e purificação do peptídeo (P10)..........................................................44

4.7 Imunização dos camundongos.........................................................................44

4.8 Grupos utilizados...............................................................................................45

4.9 Análise da carga fúngica através das unidades formadoras de colônias....45

4.10 Dosagem de citocinas do homogenato de pulmão pelo método de CBA..46

4.11 Detecção por ELISA de anticorpos (IgG Total, IgG2a, IgG2b, IgG1 e IgG3)

no soro de camundongos BALB/c anérgicos infectados intratraquealmente

com P. brasiliensis e imunizados ou não com o peptídeo P10...........................46

4.12 Dosagens de óxido nítrico...............................................................................47

4.13 Análise imunohistoquimico através da reação de estreptavidina-biotina-

peroxidase.................................................................................................................47

4.14 Ensaio de proliferação celular........................................................................48

4.15 Dosagem de citocinas do sobrenadante de cultura celular.........................49

4.16 Imunofenotipagem por citometria de fluxo....................................................50

4.17 Contagem global de leucócitos......................................................................50

4.18 Curva de sobrevida..........................................................................................51

4.18.1 Curva de sobrevida de camundongos Knockout iNOS...................................51

4.19 Histopatologia...................................................................................................51

4.20 Determinação da fibrose pulmonar................................................................52

4.21 Marcação com radiofármaco...........................................................................52

4.22 Estudios de imagem por biomarcação...........................................................53

4.23 Determinação da atividade citotóxica in vivo do peptídeo P10...................53

4.24 Determinação da atividade citotóxica in vitro do peptídeo P10..................54

4.25 Análise estatística............................................................................................54

5 RESULTADOS........................................................................................................55

5.1 Padronizações do modelo de anergia com o glicocorticóide sintético

“fosfato de dexametasona” em camundongos BALB/c.......................................55

5.2 Análise da carga fúngica através das unidades formadoras de colônias 1°

protocolo: (Os animais foram sacrificados com 45 dias de infecção)................58

5.3 Análise da carga fúngica através das unidades formadoras de colônias 2°

protocolo: (Os animais foram sacrificados com 60 dias de infecção)................60

5.4 Análise das citocinas IFN-, IL-12, TNF-α, IL-10, IL-6, IL-8, MCP-1, IL-4 e IL-2

nos camundongos anérgicos infectados e tratados com drogas antifúngicas,

associadas, ou não, à imunização com P10 pelo método de CBA......................62

5.4.1 Padrão Th1/Th2 determinado pela análise do perfil de citocinas.....................71

5.5 Detecção por ELISA de anticorpos (IgG Total, IgG2a, IgG2b e IgG1) no soro

de camundongos BALB/c anérgicos infectados intratraquealmente com P.

brasiliensis e imunizados ou não com o peptídeo P10........................................72

5.5.1 Dosagem de IgG total........................................................................................72

5.5.2 Dosagem de IgG2a...........................................................................................73

5.5.3 Dosagem de IgG2b e IgG1................................................................................74

5.6 Dosagem de óxido nítrico (NO) do homogeneizado de pulmão....................76

5.7 Dosagem de óxido nítrico (NO) do sobrenadante de cultura celular............77


peroxidase.................................................................................................................78

5.9 Ensaio de proliferação celular..........................................................................82

5.10 Dosagem de citocinas do sobrenadante de cultura celular.........................83

5.11 Caracterização de células T com fenótipo de memória................................90

5.12 Caracterização de células T com fenótipo de reguladoras (Treg)...............92

5.13 Contagem global de leucócitos......................................................................94

5.14 Curva de sobrevida de camundongos BALB/c anérgicos...........................95

5.14.1 Curva de sobrevida de camundongos Knockout iNOS...................................96

5.15 Histopatologia do pulmão de camundongos anérgicos infectados e

imunizados, ou não, com o peptídeo 10................................................................97

5.16 Determinação da fibrose pulmonar................................................................98

5.17 Biodistribuição do conjugado radiomarcado..............................................102

5.18 Aquisições de imagens cintilográficas........................................................103

5.19 Determinação da atividade citotóxica in vivo do peptídeo P10.................104

5.20 Determinação da atividade citotóxica in vitro do peptídeo P10................106

6 DISCUSSÃO.........................................................................................................108

7 CONCLUSÕES.....................................................................................................115

REFERÊNCIAS........................................................................................................117

APÊNDICES.............................................................................................................131

APÊNDICE A - Artigos publicados em periódicos indexados...........................131

APÊNDICE B - Artigos no prelo............................................................................132

APÊNDICE C - Patente...........................................................................................132

Introdução

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23

1 INTRODUÇÃO

1.1 Paracoccidioidomicose

A paracoccidioidomicose (PCM), micose sistêmica com manifestações clínicas de

uma doença granulomatosa, causada pelo fungo termodimórfico Paracoccidioides

brasiliensis, foi descrita pela primeira vez, por Adolpho Lutz, ao verificar lesões

bucais em dois pacientes, em 1908. A micose descrita por Lutz foi inicialmente

nomeada de Blastomicose Sul-Americana ou Doença de Lutz-Splendore e Almeida

(LACAZ et al., 1991), porém, a oficialização do termo Paracoccidioidomicose foi

estabelecida em 1971, em Medellín, Colômbia, durante reunião de micologistas do

continente Americano, sendo mundialmente aceita, desde então (LACAZ et al.,

1982).

A PCM é endêmica e considerada a micose sistêmica de maior prevalência na

América Latina, reportada desde o Mexico até a Argentina e afetando principalmente

Brasil com 80% dos casos seguido pela Colômbia e Venezuela (BRUMMER et al.,

1993; WANKE et al., 1994). Casos atípicos ou não autóctones foram notificados no

Chile, Suriname, Guiana, Nicarágua e Belize segundo Restrepo (20031 apud

RESTREPO et al., 2008). Um aspecto interesante na distribuição da doença é que

paises onde a PCM é endemica, não se apresenta uma distribuição homogenea da

doença e sim em certas regiões que oferecem condições aparentemente favoraveis

para o fungo (RESTREPO et al., 2001).

No Brasil, os índices de mortalidade mais elevados dessa micose, foram

encontrados nas regiões Sudeste, especialmente nos estados de São Paulo, Minas

Gerais e Rio de Janeiro e na região sur nos estados de Paraná e Rio Grande do Sul

(PRADO et al., 2009); sendo considerado um grave problema de Saúde Pública,

devido à existência de extensas áreas endêmicas, ao alto potencial incapacitante e à

quantidade de mortes prematuras que provoca, principalmente para segmentos

sociais específicos, como os trabalhadores rurais, trazendo importantes

repercussões econômico-produtivas dos indivíduos acometidos (BLOTTA et al.,

1999; SHIKANAI-YATSUDA et al., 2006).

No Brasil, a investigação epidemiológica sobre a mortalidade é realizada pelo

Sistema de Informações sobre Mortalidade (SIM), onde a taxa anual de mortalidade

1RESTREPO A. Paracoccidioidomycosis. In: DISMUKES, W. E.; PAPPAS, P. G.; SOBEL, J. (Ed.)

Clinical mycology. New York: Oxford University Press, 2003. p. 328-345.

Introdução

Muñoz J. E

24

detectada foi de 1,45/milhão de habitantes, mostrado que 94,6% das mortes

ocorreram no Sul, 90,9% no Sudeste, 87,3% no Centro-Oeste e 74,6% no Norte e

Nordeste do Brasil, onde o sub-registro é mais comum o sub-registro tem sido

essencialmente atribuído à falta de atestado de óbito padronizado, que geralmente

ocorre em áreas pobres e rurais (PRADO et al., 2009).

Epidemiologicamente apresenta incidência maior em pacientes do sexo

masculino, com idades entre 30 e 50 anos, e uma proporção de homem para mulher

de (13:1), esta proporção não se aplica na infância, onde a micose é distribuída

uniformemente entre ambos os sexos, com ligeiro predomínio do masculino em

adultos jovens (BRUMMER et al., 1993; RESTREPO; TOBON, 2005; RESTREPO et

al., 2008).

A doença no homem pode-se desenvolver desde o momento em que o fungo

entra em contato com o hospedeiro, ou passar por um período de latência que pode

ser de meses a vários anos, dificultando assim, a determinação precisa do local

onde foi adquirida a infecção, o P. brasiliensis também tem a capacidade de

permanecer latente no pulmão por um período de tempo indefinido (BRUMMER et

al., 1993; RESTREPO, 1994).

Deve-se salientar que a PCM apresenta maior incidência em populações com

baixos recursos econômicos, que não têm acesso aos serviços de saúde,

principalmente aqueles que desempenham atividades na lavoura (MARQUES et al.,

1983). Relatos de pacientes com PCM recidentes em grandes centros urbanos ou

paises não endêmicos como EUA, África, Europa e Ásia são conhecidos como

doença de importação, devido ao fato dos pacientes terem residido em regiões

endêmicas em época anterior à manifestação clínica da micose, e nestes grandes

centros a doença é diagnosticada, um estudo realizado por Forjaz (1989), indicou o

fluxo migratório de zonas endêmicas do Brasil à cidade de São Paulo, e foi

observado que 80,8% dos pacientes tinham desempenhado atividades na lavoura e

67,7% proveniam de outros estados.

Diversas opções têm sido publicadas para clasificar as formas clínicas da

PCM, baseadas em diferentes critérios tais como topografia das lessões, gravidade

da doença, resultados de reações sorológicas e história natural, entre outras.

Algumas adaptações da clasificação apresentada no International Colloquium on

Paracoccidioidomycosis, clasificam a doença em: PCM infecção, PCM doença,

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25

Forma aguda/subaguda, Forma crônica, Forma unifocal, Forma multifocal e a forma

residual ou sequelar. A classificação mais sitada atualmente da doença baseia-se

em duas formas clínicas principais, a aguda e subaguda (tipo juvenil) e a crônica

(tipo adulto) é determinada por uma série de sinais e sintomas (FRANCO et al.,

1987), dependentes da evolução, da localização das lesões e da ativação do

sistema imune do hospedeiro (MONTENEGRO; FRANCO, 1994).

Pacientes com a forma crônica da PCM, que compreendem aproximadamente

90% dos casos, apresenta uma duração prolongada com uma instalação lenta e

gradual, tendendo a se localizar em órgãos e tecidos de modo mais focal,

comprometendo principalmente o pulmão e a mucosa das vias respiratórias, com

manutenção da resposta imune celular e baixos títulos de anticorpos específicos, o

que corresponde à forma hiperérgica ou localizada (MENDES; RAPHAEL, 1971;

MONTENEGRO; FRANCO, 1994). A forma crônica progride lentamente, de forma

silenciosa e é denominada unifocal quando é restrita a um órgão, geralmente a

doença envolve vários órgãos (forma multifocal), sendo os pulmões (90% dos

pacientes), mucosa e pele os sítios mais acometidos pela infecção (SHIKANAI-

YATSUDA et al., 2006).

Enquanto que as formas agudas e subagudas apresentam altos níveis de

anticorpos específicos e o sistema imune celular comprometido. A resposta imune

desenvolvida por pacientes com a forma aguda e subaguda leva à formação de

granulomas frouxos, que estão associados ao alto número de células fúngicas

viáveis (FRANCO et al., 1987).

A forma aguda apresenta evolução rápida, e é responsável por 15 a 20% de

todos os casos da doença, com frecuente linfadenomegalia, hepatoesplenomegalia,

envolvimento ósteo-articular, mediastinal, intra-abdominal e lesões cutâneas

(SHIKANAI-YATSUDA et al., 2006), apresentando lesões progressivas e afetando o

sistema fagocítico mononuclear de jovens de ambos os sexos e pessoas

imunocomprometidas, apresentando ativação policlonal de linfócitos B por

antigenemia, que corresponde à forma anérgica ou disseminada (CAMARGO;

FRANCO, 2000; MONTENEGRO; FRANCO, 1994).

O estabelecimento da infecção parece estar intimamente relacionado ao

dimorfismo do P. brasiliensis, conforme observado em outros fungos patogênicos

(Histoplasma capsulatum, Blastomyces dermatitidis, Sporothrix schenckii e

Introdução

Muñoz J. E

26

Coccidioides immitis). Essa conversão considera-se fundamental para o

estabelecimento da infecção e de interesse particular, pois se acredita que seja

importante na patogenicidade do fungo (CAMARGO; FRANCO, 2000; FRANCO,

1987; SAN-BLAS et al., 2000). A primeira interação fungo-hospedeiro ocorre nos

alvéolos pumonares, onde dependendo da capacidade invasiva do fungo e do

estado imunológico do hospedeiro, começa o processo de dimorfismo e colonização.

Estudos realizados no modelo experimental de infecção com conídios de P.

brasiliensis, descrivem que o processo de dimorfismo ocorre entre 12 e 18 horas

após infecção (MCEWEN et al., 1987).

A mulher está mais protegida da doença, devido à presença de estrógenos

endógenos que atuam a través de proteínas ligantes no citosol do fungo, inibindo a

transformação de micélio em levedura (STOVER et al., 1986). Assim, sem a

interferência hormonal, a infecção pode aparecer antes da puberdade ou depois da

menopausa, uma vez que o fungo P. brasiliensis tem receptores para 17-β-estradiol

(E2) no citoplasma.

Aristizabal et al (2002), avaliaram o curso clínico da doença no modelo

experimental de camundongos machos e fêmeas, eles empregaram diferentes

condições hormonais e observaram que os machos que receberam o E2

conseguiram restringir a progressão da doença, enquanto que as fêmeas que

receberam testosterona não foram capazes de controlar a doença.

No envolvimento pulmonar é importante resaltar as lesões fibróticas residuais,

observadas em ~60% dos pacientes com PCM, esta é uma severa e progressiva

sequela que altera as funções respiratórias e incapacita o paciente nas suas

atividades diárias tornando-se um problema econômico-productivo (NARANJO et al.,

2011; TOBÓN et al., 2003).

Interpretações de estudos de função pulmonar têm sido dificultadas pela alta

freqüência de uso do tabaco entre os doentes de PCM, qual exede o 93% dos

pacientes, apresentando defeito predominante obstrutivo, principalmente das

pequenas vias respiratórias, com hipoxemia de grau variado. Além disso, o fumo

parece ser um factor de risco para o desenvolvimento da micose (LEMLE et al.,

1983).

Casos de PCM em pacientes imunossuprimidos não são maioria, já que o P.

brasiliensis não é um fungo “oportunista”, mas alguns casos de infecção associados

Introdução

Muñoz J. E

27

a imunossupressão tem sido relatados, como por exemplo em pacientes submetidos

a tratamento citotóxico ou terapia imunossupressora contra o câncer (MARQUES;

LASTÓRIA; MARQUES, 2011), pacientes submetidos a transplantes (SHIKANAI–

YASUDA et al., 1995; SUGAR et al., 1984), desordens imunológicos como Lúpus

eritematoso (LONDERO; SANTOS; RAMOS, 1987), artrite reumatoide

(WOYCIECHOWSKY et al., 2011) e doença de Hodgkin (ALTERIO; NEGRO, 1960).

Presume-se que fármacos citotóxicos pode reativar uma lesão latente, causando

uma infecção pulmonar aguda, provavelmente pelo comprometimento do sistema

imunológico devido ao tratamento quimio e radioterápico.

1.2 Agente etiológico

P. brasiliensis, agente etiológico da PCM, apresenta dimorfismo térmico,

assim, à temperatura ambiente, entre 18-25 oC, assume forma de micélio, composto

por hifas septadas que produzem propágulos asexuais, conocidos como

microconideos (˂ 5 µm). Já no hospedeiro ou em cultura a uma temperatura entre

35-37 oC, apresenta a forma de levedura unicelular, com múltiplos brotamentos

(blastoconídeos) e tamanho variável entre 6 a 40 µm (RESTREPO et al., 2008). Na

fase de levedura, as colônias são enrugadas e com coloração creme; o seu

crescimento começa aparentemente entre 10 a 15 dias após a incubação (LACAS et

al., 1991). Durante vários anos, o P. brasiliensis tem sido conhecido apenas na sua

forma assexuada, atualmente sabe-se que, pelo menos, uma quinta parte dos

fungos descritos não é conhecida a sua fase sexual (GEISER; PITT; TAYLOR,

1998). No entanto, a incapacidade para demonstrar a fase sexual, não significa a

sua inexistência. Recentemente, genomas sequenciados e alguns dados mostram

evidências de recombinação, corroborando a existência de uma fase sexual do P.

brasiliensis (MATUTE et al., 2006).

A infecção produzida nos humanos por P. brasiliensis acontece

principalmente pela inalação dos conídios ou propágulos na forma miceliana do

fungo; com o aumento da temperatura, os conídios recebem um estímulo e se

transformam em leveduras nos alvéolos pulmonares, constituindo a forma parasitária

nos tecidos do hospedeiro, como foi comprovado experimentalmente em

camundongos (McEWEN et al., 1987). Até recentemente, os humanos eram tidos

Introdução

Muñoz J. E

28

como os únicos hospedeiros naturalmente infectados por este fungo, mas é

importante resaltar que alguns animais foram encontrados portadores da infecção,

como tatus especialmente Dasypus novemcinctus (BAGAGLI et al., 1998.,

CORREDOR et al., 1999; NAIFF et al., 1986; SILVA-VERGARA et al., 2000),

cachorros (FARIAS et al., 2005, 2011; RICCI et al., 2004), morcegos frugívoros

Artibeus lituratus (GROSE; TAMSITT, 1965), bicho-preguiça Choloepus didactylus

(TREJO-CHAVES et al., 2011), espécies de macacos (JOHNSON; LANG 1984;

NAIFF et al., 1996) e o fungo também foi encontrado em feces de pinguins da

antartida (GARCIA et al., 1993; GEZUELE, 1989) provavelmente, estes últimos

animais foram infectados a partir de solos contaminados com o fungo (FRANCO et

al., 2000).

Após a descrição do fungo P. brasiliensis por Adolpho Lutz, em 1908, iniciou-

se uma fase de estudos das principais características do agente infectante, como

diferenças morfológicas e temperatura de cultivo. Splendore, em 1912, sugeriu a

classificação do agente no gênero Zymonema, recebendo, assim, a denominação de

Zymonema brasiliense. A doença passou, então, a ser denominada de

"blastomicose brasileira" e, logo a seguir, "blastomicose sul-americana", já que foram

relatados casos isolados em outros países da América do Sul. Após estudos

sistematizados, cria-se um novo gênero dentro do reino Fungi - o Paracoccidioides,

revalidando o nome da espécie criada por Splendore, em 1912. Em 1930, Floriano

Paulo de Almeida oficializou o nome Paracoccidioides brasiliensis (ALMEIDA, 1930).

Taxonomicamente, o fungo P. brasiliensis foi, inicialmente, classificado por Ajello

(1977) da seguinte forma: Reino Fungi, Filo Eumycota, Subdivisão Deuteromycotina,

Classe Hyphomycetes, Ordem Moniales, Família Moniliaceae, Gênero

Paracoccidioides e Espécie brasiliensis. Contudo, outra classificação foi proposta,

pois estudos filogenéticos empregando ferramentas moleculares posicionaram o

agente etiológico da PCM junto aos demais fungos dimórficos (Coccidioides

posadasii, Coccidioides immitis, Blastomyces dermatitidis e Histoplasma capsulatum)

como pertencentes à seguinte categoria taxonômica: Reino Fungi, Filo Ascomycota,

Classe Pleomycetes, Ordem Onigenales, Família Onygenaceae, Gênero

Ajellomyces e Espécie brasiliensis, sendo denominado, então, de Paracoccidioides

brasiliensis (SAN-BLAS; NINO-VEGA; ITURRIAGA, 2002).

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O habitat natural do fungo não tem sido precisamente definido, o solo é

considerado o habitat do P. brasiliensis apesar de seu isolamento ser raro (FRANCO

et al., 2000). Na intenção de identificar o micronicho de fungo tem sido prejudicado

por várias circunstâncias, incluindo a falta de informações sobre focos que poderiam

ajudar a determinar uma fonte comum de infecção, além disso, o fungo tem a

capacidade de entrar em prolongados estágios de repouso, como tem sido

observado em casos relatados em indivíduos que vivem em países não endêmicos

que muitos anos antes viveram em uma área endêmica.

Há poucos dados a respeito da forma em que o fungo P. brasiliensis

encontra-se na natureza (RESTREPO, 1985); entretanto, Negroni (1966) e Albornoz

(1971) isolaram o fungo P. brasiliensis do solo. Fatores ecológicos também foram

demonstrados em municípios da Colômbia onde pacientes tinham vivido durante

muitos anos, encontrando uma correlação com residência entre 1000 a 1499 m

acima do nível do mar, com uma alta taxa de precipitação anual (2000 a 2999 mm) e

com culturas agrícolas como café e tabaco (CALLE et al., 2001; TORRADO et al.,

2000). A partir de então, estudos a respeito do habitat do fungo começaram a ser

realizados. Silva-Vergara et al. (1998) isolaram o fungo a partir de solo de regiões

com plantação de café no Estado de Minas Gerais, sugerindo que esse é um dos

habitats do fungo P. brasiliensis, reafirmando, assim, a hipótese da PCM ser comum

nas áreas agrícolas.

1.3 Fatores de virulência

Os fatores de virulência auxiliam na aderência, colonização, disseminação e

habilidade do fungo para sobreviver a ambientes hostis e escapar dos mecanismos

da resposta imune do hospedeiro. Tanto os fatores de virulência apresentados por

diferentes fungos, como os mecanismos de defesa do hospedeiro requerem ação e

interação de processos complexos, cujo conhecimento permitirá a melhor

compreensão da patogenia das micoses sistêmicas (KUROKAWA, 1998).

Pouco se conhece sobre os mecanismos de virulência que permitem ao fungo

adaptar-se às condições presentes no tecido do hospedeiro, para iniciar a

colonização e proceder à invasão (LACAZ et al., 2002; RESTREPO; BENARD 2004;

SAN-BLAS et al., 2002). Não obstante, podemos assumir que o dimorfismo é um

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30

fator de virulência importante, já que em sua ausência não se daria a conversão dos

conídios em leveduras no pulmão, interrompendo-se assim a infecção. A conversão

dimórfica é a regra em modelos experimentais, por isso, seu significado na

patogênese da PCM é claro (MENDES-GIANNINI et al., 2000; RESTREPO-

MORENO, 2003).

A parede celular dos microrganismos desperta grande interesse e concentra

muitos estudos acerca de sua biossíntese e composição, pois representa uma

barreira de proteção contra processos físico-químicos, aos quais, a célula fúngica

está sujeita, e por ser, também, o ponto de contato para adesão e subseqüente

invasão do tecido hospedeiro (REISS et al., 1992).

A composição de polissacarídeos da parede celular de P. brasiliensis pode

estar em parte, relacionada à sua virulência. Estudos bioquímicos sobre os

polissacarídeos da parede celular do fungo na forma filamentosa e de levedura

mostraram que os principais constituintes são: glucanas, quitina, proteínas e lipídios

(KANETSUNA et al., 1969, 1972; KANETSUNA, CARBONELL, 1970).

A virulência do P. brasiliensis tem sido associada à -1,3 glucana contida na

parede da célula leveduriforme do fungo (MORAES; SCHAFFER, 2002). Estudos

têm mostrado que α-glucana é o principal polissacarídeo da parede de leveduras,

formas estas que apresentam apenas traços de β-glucanas. Já na parede do

micelio, a β-glucana é a única glucana presente. Isso tem suscitado a hipótese de

que a transformação dimórfica de P. brasiliensis exija controle rigoroso da síntese

de glucana (BRUMMER; CASTANEDA; RESTREPO, 1993).

San-Blas et al. (1984) demonstraram alteração na síntese de -1,3 glucana

quando amostras passaram por repiques sucessivos (in-vitro), ocasionando a perda

ou a atenuação da sua virulência. Os mesmos autores verificaram, entretanto, que a

adição de soro fetal bovino à cultura induz a produção desse polissacarídeo, com

conseqüente reativação da virulência. Entretanto, um estudo que compara três

isolados distintos de P. brasiliensis (Pb192, Pb18 e Pb265) mostrou uma contradição

à observação e demonstrou que a virulência das células leveduriformes do fungo P.

brasiliensis não esteve correlacionado com os níveis de -1,3 glucana na parede

celular (ZACHARIAS et al., 1986).

Estudos realizados in-vitro com isolados de P. brasiliensis sugerem que -1,3

glucana protege o fungo contra enzimas digestivas do hospedeiro como também

Introdução

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31

leucócitos e macrófagos (SAN-BLAS, G., SAN-BLAS, F, 1977). Brummer et al.

(1990) observaram, também, que a virulência de isolados de P. brasiliensis era

atenuada ou perdida quando este era mantido por muito tempo in-vitro.

Componentes da parede celular de P. brasiliensis, como ß-glucana,

estimulam a resposta imune em uma intensidade mais alta ou mais reduzida. Uma

resposta imune intensa permitiria uma menor sobrevivência das células fúngicas,

prevenindo a instalação e o crescimento do patógeno. Assim, a ß-glucana presente

na parede celular do fungo P. brasiliensis é capaz de induzir uma resposta

inflamatória mais vigorosa e de produzir o fator de necrose tumoral (TNF), uma

citocina importante que estimula a atividade fungicida dos macrófagos

(FIGUEIREDO et al., 1993; SILVA; ALVES; FIGUEIREDO, 1994).

Gómez et al. (2001) demonstraram que o P. brasiliensis é capaz de sintetizar

melanina in vitro e durante a infecção. Conídios e células leveduriformes cultivadas

em meio contendo L-DOPA foram reativas com anticorpos monoclonais que

reconhecem a melanina. Através de camundongos infectados com conídios de P.

brasiliensis, foram recuperados resíduos escuros reativos com anticorpos

monoclonais contra melanina (NOSANCHUK et al., 1999).

Por métodos físico-químicos e imunológicos mostrou-se que os conídios e

leveduras podem produzir ou sintetisar, compostos melânicos, tanto in-vivo como in

vitro (GÓMEZ et al., 2001). Com base no reconhecimento da melanina como fator de

virulência, tem sido demostrado que esse pigmento pode ter um papel importante na

patogênese da PCM (GÓMEZ et al., 2001). A produção de melanina pelo fungo P.

brasiliensis parece contribuir para a virulência do fungo, pela redução da fagocitose

das células leveduriformes por linhagens de macrófagos peritoneais, alveolares e

primários (J774.16 e MH-S), aumentando a resistência do patógeno contra o ataque

dessas células efetoras. As células melanizadas do fungo são também menos

susceptíveis a drogas antifúngicas, particularmente à anfotericina B (DA SILVA et

al., 2006).

A análise por SDS-PAGE do material eluído do sobrenadante de cultura do P.

brasiliensis revelou quatro componentes de 43, 55, 70 kDa e um de alto peso

molecular, com migração difusa. Em muitos casos estos componentes glicoproteicos

induzem uma resposta imune baseada na secreção de anticorpos a qual não é a

mais eficiente, embora tenha sido demonstrado a importância de alguns anticorpos

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32

monoclonais na resposta a algumas doenças fúngicas como a PCM (BUISSA-FILHO

et al., 2008) (Apêndice 3). Além de ser fundamental no diagnóstico da PCM, a gp43

tem sido assinalada como fator de virulência, demonstrando se ligar especificamente

na laminina, proteína da matriz extracelular. A ligação à laminina induz um aumento

na adesão do fungo às células epiteliais (VICENTINI et al., 1994). Além disso, foi

observado que células mononucleares de sangue periférico (PBMC) de pacientes

com PCM quando estimuladas com gp43, aumentam a secreção de IL-10, uma

importante citocina imunossupressora capaz inibir a expressão de citocinas

proinflamatorias. Nesse mesmo estudo, foi observado um aumento na taxa de

apoptose nos PBMC estimuladas com esta glicoproteina (CACERE et al., 2002).

1.4 Paracoccidioidomicose e o sistema imune

O estabelecimento da doença, sua disseminação e gravidade além de estarem

relacionado ao próprio fungo, como sua virulência, composição antigênica e até

condições ambientais, dependem muito dos fatores ligados ao próprio hospedeiro na

sua capacidade de desenvolver ou não uma resposta imune eficaz.

Células da resposta imune inata como células natural killer (NK), neutrófilos,

monócitos, macrófagos e células dendríticas, desempenham um papel central na

resistência, já que constituem a primeira linha de defesa a entrar em contato com o

P. brasiliensis assim que ele se instala nos pulmões. A participação destas células

na reação inflamatória e na atividade fungicida é induzida pelo fungo e por citocinas

produzidas pelas células durante sua interação com fagócitos (CALVI et al., 2003).

As células NK foram estudadas no sangue periférico de pacientes com a PCM

e no modelo experimental em hamster. Estas células apresentam atividade

citotóxica diminuída quando se tem a doença, sugerindo distúrbio imunológico

associado à depressão da imunidade celular tanto em pacientes quanto no modelo

experimental (PERAÇOLI et al., 1995).

Os neutrófilos exercem um importante papel imunoprotetor na fase inicial da

defesa, mas deve-se resaltar que dependendo dos padrões genéticos do hospedeiro

podem apresentar um papel imunoprotetor ou imunoregulador. Em camundongos

suscetíveis à PCM (B10.A), a depleção dos neutrófilos, resultou em um aumento nos

níveis de anticorpos IgG1, IgA e IgG3 relacionados com a secreção de IL-4, TGF-β e

Introdução

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IFN-γ, respectivamente. Já em animais resistentes (A/J), houve a produção de altos

títulos de IgG2a, IgG3 e IgG2b os quais são isotipos Th1 ou IFN-reguladores, desta

forma os neutrófilos são fundamentais em situações onde a imunidade mediada por

células esta debilitada (PINA et al., 2006).

Outras células que desempenham um papel importante na resposta à infecção

por P. brasiliensis são os monócitos e macrófagos (Mø), sendo a ativação destas

células um dos primeiros eventos da resposta imune inata para combatir o fungo.

Esta ativação é induzida pelo reconhecimento de componentes extracelulares do

fungo por alguns dos receptores de reconhecimento padrão (PRRs) (NAKAIRA et

al., 2011). Também é possivél ativar os Mø por ação do IFN-γ, o qual induz um

aumento na produção de óxido nítrico (NO) e outros óxidos nitrogenados induzidos

pela NO sintase induzível (iNOS), potencializando assim a atividade microbicida dos

Mø. O papel do NO no controle da infecção por P. brasiliensis é importante tanto na

fase micelial (GONZALEZ et al., 2000) quanto na fase leveduriforme no modelo in

vivo (BOCCA et al.,1998). No entanto o papel do NO na PCM permanece

controverso, já que a produção excessiva de NO pode estar relacionada com

suceptibilidade no proceso de infecção (NASCIMENTO et al., 2002).

As células dendríticas (DCs) são as pricipais células apresentadoras de

antígeno e recentes estudos experimentais têm demonstrado que a infecção com o

P. brasiliensis ativa estas células a migrar para a região dos nódulos linfáticos,

providenciando o primeiro contato para a posterior ativação de uma resposta do tipo

Th1 (SILVANA DOS SANTOS; FERREIRA; ALMEIDA, 2011).

Dados clínicos e experimentais indicam que a imunidade mediada por células

desempenha um papel essencial na defesa do hospedeiro contra a infecção por P.

brasiliensis, ao passo que níveis elevados de anticorpos específicos e a ativação

policlonal das células B estão associados com as formas mais graves da doença

(CANO et al., 1998).

Subpopulações de células T auxiliares são conhecidos por estimular diferentes

vias de resposta imune (CHERWINSKI et al., 1987). A imunidade contra o P.

brasiliensis pode ser regulada por subconjuntos Th1 ou Th2, o que determina o

resultado da infecção. Linfocinas produzidas pelos linfócitos Th1 estão

correlacionadas com a resistência contra certas doenças entre elas a PCM

(NEGRONI, 1993).

Introdução

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34

A persistência a longo prazo de patógenos em um hospedeiro são

características de certas doenças infecciosas, entre elas a esquistossomose,

leishmaniose e paracoccidioidomicose (PCM). Algumas células do sistema imune

podem ser utilizadas pelos microrganismos para permanecer no hospedeiro, como é

o caso das células Treg conhecidas por suprimir a ativação e funções efectoras das

células T (BELKAID; ROUSE, 2005).

Estudos de imunoprecipitação com soros de pacientes com PCM

demonstraram que a glicoproteína de 43 kDa (gp43) era constantemente

imunoprecipitada no soro dos pacientes com PCM e não era reativa com soro de

indivíduos normais (PUCCIA; TAKAOKA; TRAVASSOS, 1991). Assim, a gp43 tem

sido vista como o principal marcador sorológico da PCM, aumentando a

especificidade e a sensibilidade dos testes sorológicos (CAMARGO et al., 1994;

TABORDA; CAMARGO, 1993, 1994). A gp43 contém epítopos com a capacidade de

produzir uma resposta imune celular (DTH) em cobaias (RODRIGUES;

TRAVASSOS, 1994) e em pacientes humanos (SARAIVA et al., 1996). A

sensibilidade da resposta imune em camundongos pela gp43 ocorre pela

proliferação de CD4+ (TRAVASSOS et al., 1995). Estes epítopos estimulam os

linfócitos CD4+ Th1, os quais produzem interferon (IFN-ᵧ), que tem a função de

estimular à formação de granulomas que podem conter as leveduras (BRUMMER et

al., 1988). Entretanto, a contribuição de cada subtipo de celulas T na resposta imune

depende dos padrões genéticos do hospedeiro, e uma imunidade com um balanço

das células T CD4/CD8 regula a secreção de citocinas do tipo Th1 e Th2, o que se

correlaciona com a resistência do hospedeiro à infeção por P. brasiliensis

(CHIARELLA et al., 2007).

A gp43 é expressa de forma constitutiva na fase micelial e leveduriforme do

fungo (GOLDANI et al., 1994), sendo constituída por uma única cadeia de

oligossacarídeos (ALMEIDA et al., 1996) e caracterizada bioquimicamente por

Puccia et al. (1986).

A glicoproteína de 43 kDa (gp43) possui 416 aminoácidos, onde um trecho

especifico de 15 aminoácidos designado como P10, é reconhecido pelos linfócitos T

de camundongos e humanos. O efeito protetor do P10 esta relacionado em induzir

uma resposta imune do tipo Th1 dependente de IFN-γ- dependente em

camundongos isogênicos (TABORDA et al., 1998).

Introdução

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Camundongos de linhagens isogênicas, ao serem imunizados com P10,

desenvolveram infecção pulmonar 200 vezes menos intensa do que os animais não

imunizados (TABORDA et al., 1998). Iwai et al. (2003) e a análise através do

programa TEPITOPE, que verifica a probabilidade de HLA-DR de caucasianos

reconhecerem diferentes peptídeos, demonstrou que o P10 é um peptídeo

promíscuo, sendo um importante candidato vacinal para ser utilizado em humanos.

Este peptídeo, quando associado às drogas comumente utilizadas no

tratamento da paracoccidioidomicose, apresenta um efeito aditivo no modelo

experimental utilizando camundongos BALB/c, o que demonstra a capacidade do

peptídeo P10 em auxiliar na diminuição do tempo de tratamento desta micose

(MARQUES et al., 2006).

1.5 Dexametasona e resposta imune

A dexametasona (Dex) é um potente corticosteróide sintético que possui baixo

custo e é amplamente utilizada com propriedades imunossupressoras e

antiinflamatórias (RHODUS et al., 2006). Os corticóides são hormônios esteróides

que atuam nos processos alérgicos, inflamatórios, imunológicos e no metabolismo

de carboidratos, proteínas e gorduras (DIASIO; LOBUGLIO, 1996). A Dex possui

mecanismos de ação idênticos ao do cortisol (ou hidrocortisona), que é o mais

importante corticosteróide natural produzido pelo córtex da glândula supra-renal. A

Dex, quando comparada ao cortisol, apresenta as seguintes propriedades: maior

potência anti-inflamatória, meia-vida e tempo de ação prolongado, redução dos

efeitos de retenção de sal, atividade tópica e oral (KATZUNG; TREVOR, 1995). Um

dos mecanismos de supressão da dexametasona é o bloqueio da expressão do

Nuclear Factor-KappaB (NF-KB) e a diminuição nos níveis de citocinas

proinflamatorias como IL-8, IL-6 e IL-1β. Estes corticoides são conhecidos por

diminuir a inflamação através da diminuição do influxo de células inflamatórias.

Seus efeitos na inibição da inflamação são profundos e rápidos, entre eles, a

diminuição da fase vascular da inflamação, através de vasoconstrição; diminuição da

permeabilidade capilar; menor liberação de cininas vasoativas e queda da histamina

tecidual. Promove também a indução de um menor afluxo de eosinófilos e inibição

Introdução

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da função dos macrófagos, um dos efeitos mais importantes e mais citados

(BAXTER; FORSHAM, 1972; CLAMAN, 1975).

1.6 Tratamento da Paracoccidioidomicose

A micose quando não é diagnosticada e tratada oportunamente, pode levar as

formas disseminadas graves e letais, com rápido e progressivo envolvimento dos

pulmões, tegumento, gânglios, baço, fígado e órgãos linfóides do tubo digestivo

(SHIKANAI-YASUDA et al., 2006).

O tratamento da PCM é muito difícil, exigindo uma terapia prolongada para

obter um resultado bem sucedido (RESTREPO, 1994). Apesar da limitação das

informações disponíveis em estudos comparativos com diferentes esquemas

terapêuticos, sugere-se o itraconazol como a opção terapêutica. Entretanto,

considerando que o medicamento não está disponível na rede pública da maioria

dos estados, a combinação sulfametoxazol-trimetoprim é a alternativa mais utilizada

na terapêutica ambulatorial dos pacientes com PCM. Pacientes com formas graves,

necessitando internação hospitalar, devem receber anfotericina B ou associação

sulfametoxazol/trimetoprim por via intravenosa. A duração do tratamento relaciona-

se à gravidade da doença e ao tipo de droga utilizada. Usualmente, o tratamento é

de longa duração, para permitir o controle das manifestações clínicas da micose e

evitar as recaídas. O paciente deve permanecer em tratamento e acompanhamento

até a obtenção dos critérios de cura, com base nos parâmetros clínicos, radiológicos

e sorológicos (SHIKANAI-YASUDA et al., 2006).

1.6.1 Derivados azólicos

Os antifúngicos azólicos foram descobertos na década de 1960 e são

totalmente sintéticos, sendo dividido em duas classes segundo sua estrutura:

imidazóis (duas moléculas de nitrogênio) e triazóis (três moléculas de nitrogênio).

Entre os imidazóis encontramos o miconazol e o cetoconazol, este último com

atividade sistêmica. Contudo todos os triazóis possuem atividade sistêmica,

incluindo o fluconazol, itraconazol e voriconazol. Tanto os imidazóis como os triazóis

inibem a biossíntese do ergosterol pela inibição da enzima 14-α-desmetilase do

Introdução

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37

citocromo P-450 que é responsável pela demetilação do lanosterol. No entanto, os

triazóis apresentam uma maior seletividade pelo sítio de ação em relação aos

imidazóis, devido ao acréscimo de nitrogênio em seu anel azóico

(GEORGOPAPADAKOU, 1998). Geralmente o mais utilizado destes azóis no

tratamento da PCM é o itraconazol o qual é sugerido como uma opção terapêutica

que permitiria o controle das formas leves e moderadas da doença em menor

período de tempo, sendo utilizado na concentração de 200 mg/dia para adultos

durante um período de 6 a 9 meses nas formas leves da doença e de 12 a 18 meses

nas formas moderadas (SHIKANAI-YASUDA et al., 2006). O itraconazol é

considerado um dos antifúngicos mais eficientes para combatir a PCM, com uma

eficiência do 95% dos casos e associado com baixas porcentagens de recidivas.

1.6.2 Derivados sulfonamídicos

Estes derivados sulfonamídicos foram as primeiras drogas empregadas no

tratamento da paracoccidioidomicose (SHIKANAI-YATSUDA, 2005). Entre os

derivados sulfonamídicos, a sulfadiazina é um dos principais compostos ativos (DEL

NEGRO, 1982, 1985). Ocasionalmente o P. brasiliensis pode desenvolver

resistência a esta droga durante o tratamento (RESTREPO; ARANGO, 1980). A

combinação sulfametoxazol-trimetoprim é uma opção útil e tem sido utilizada com

melhores resultados. A dose recomendada é de 1200 mg de sulfametoxazol e de

160 a 240 mg por dia de trimetoprim, durante 12 meses para formas leves da

doença e de 18 a 24 meses em infecções clínicas moderadas (SHIKANAI-YASUDA

et al., 2002). Esta associação sulfametoxazol-trimetoprim (5:1), também conhecida

como cotrimoxazol, potencializa a atividade de cada um destes antibióticos por

separado, inibindo a síntese do tetrahidrofolato e impedindo que, os microrganismos

não possam sintetizar purinas (BRUMFITT; HAMILTON-MILLER, 1993).

1.6.3 Polienos

Nos casos mais graves, a anfotericina B, um polieno, é a droga de escolha,

administrada numa dose de 0,75 mg/kg por dia, a dose cumulativa total deve ser

equivalente a 30 mg/kg. Dependendo da dose, a anfotericina B pode ter uma ação

Introdução

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fungistática ou fungicida. Este droga atua através da permeabilização da membrana

plasmática, mas sua atividade, inicialmente, depende de sua ligação ao esterol da

membrana. Essa ligação pode resultar na perda de íons intracelulares, o que

caracteriza a natureza fungistática da droga. A anfoterecina B em concentrações

fungicidas pode levar a formação de poros na membrana, com conseqüente perda

de constituintes celulares de baixo peso molecular. Essa condição é irreversível

(BRAJTBURG et al., 1990).

Justificativa

Muñoz J. E

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2 JUSTIFICATIVA

Com o propósito de diminuir os efeitos colaterais e ativar uma resposta imune

prolongada o uso de vacinas preventivas ou terapêuticas em doenças infecciosas

tem se expandido e exige a seleção de componentes imunogênicos que possam

levar a proteção do hospedeiro. No caso de doenças fúngicas, antígenos de H.

capsulatum, P. brasiliensis, B. dermatitidis, C. neoformans, C. immitis e C. albicans

estão sendo estudadas com resultados promissores (DIXON et al., 1998; JIANG et

al., 1999). O conhecimento recente de aspectos estruturais de proteínas e peptídeos

permitiu a definição de seqüências reativas e a importância de determinados

aminoácidos. De modo particular peptídeos são apresentados por moléculas de

histocompatibilidade participando em complexos que determinam o reconhecimento

de sequências “self” e “nonself” para efeito de respostas imunes ativas ou de

tolerância imunológica. Tais peptídeos têm sido explorados largamente na

constituição de vacinas juntamente aos adjuvantes apropriados.

Nossos dados prévios são promissores, portanto um melhor entendimento em

relação ao processo inflamatório pulmonar local em animais imunizados com P10 se

torna necessário, pois este peptídeo “promíscuo” é um forte candidato vacinal. Além

disso, nossa proposta é também avaliar a utilização do P10 num modelo

experimental que mimetize um processo de anergia, comum em pacientes com as

formas agudas e subagudas, e como se comportam os animais imunossuprimidos

com dexametasona, infectados e tratados com sulfametoxazol/trimetoprim ou

itraconazol, associados ou não à imunização com P10.

Objetivos

Muñoz J. E

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3 OBJETIVOS

3.1 Objetivo geral

Avaliar o papel do peptídeo 10 (P10) associado ao tratamento quimioterápico

em camundongos BALB/c anérgicos infectados com o Paracoccidioides brasiliensis.

3.2 Objetivos específicos

Padronizar um modelo experimental de anergia em camundongos BALB/c

infectados intratraquealmente com isolado Pb18;

verificar se o tratamento com antifúngicos associado à imunização com P10,

em animais imunossuprimidos, reduzirá a quantidade de fungos em

diferentes órgãos e/ou impedirá a disseminação para outros sítios

anatômicos;

verificar a produção de citocinas pró-inflamatórias nos pulmões dos animais

infectados e imunossuprimidos, submetidos ou não ao tratamento com

antifúngicos associado à imunização com P10;

verificar a produção de óxido nítrico no homogeneizado de pulmão e no

sobrenadante de cultura celular dos animais que foram imunizados com o

peptídeo P10;

identificar a geração de células T de memória durante o processo de

imunização dos camundongos;

identificar a geração de células T regulatórias durante o processo de

imunização dos camundongos.

Materiais e Métodos

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4 MATERIAIS E MÉTODOS

4.1 Animais

Foram utilizados camundongos machos da linhagem BALB/c, com a média de

idade entre 6 e 8 semanas e com um peso entre 25 e 30 gramas. Os animais foram

criados em condições SPF (Specific Pathogen Free), no biotério de camundongos

isogênicos do Departamento de Imunologia do Instituto de Ciências Biomédicas, e

mantidos no biotério de animais de experimentação do Departamento de

Microbiologia do Instituto de Ciências Biomédicas II da Universidade de São Paulo.

4.2 Imunossupressão dos Camundongos BALB/c

Os animais foram submetidos à imunossupressão com dosagens diárias de

0,15 mg/kg de fosfato de dexametasona, por um período de 20 dias. Os animais

foram mantidos em gaiolas fechadas com filtros, tentando reproduzir um ambiente

totalmente estéril, autoclavando-se a maravalha, a alimentação e a água distribuída

para os animais. As trocas das gaiolas foram realizadas duas vezes por semana em

fluxo laminar. Os animais foram considerados anérgicos quando não mais

responderam ao teste de hipersensibilidade tardia (HTT), realizado no coxim plantar

da pata traseira, utilizando-se uma solução de exoantígenos.

4.3 Avaliação do estado de imunossupressão

4.3.1 Leucograma

Com o propósito de examinar o número de leucócitos, foram realizados

leucogramas, nos quais foram coletados 20 µl de sangue (diluição 1:20) e

posteriormente adicionados a 380 µl do reagente de Turk (15 mL (ácido acético

glacial); 0,002 g (violeta de genciana); 500 mL (H2O deionizada q.s.p)), após

misturar levemente a contagem global de células foi feita em câmara de Neubauer.


Muñoz J. E

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4.3.2 Reação de HTT

Para verificar a integridade da resposta imune-celular, reações de HTT contra

o antígeno bruto de Pb 18 foram avaliadas durante a administração da

dexametasona. No coxim plantar da pata esquerda dos camundongos, foram

injetados 25 µl de solução do antígeno bruto, pela via subcutânea. A espessura da

pata dos animais foi medida 24 h após a injeção utilizando-se calibrador visual

(precisão de 0,01 mm, Mitutoyo, Japan). As medições foram diárias e as alterações

na espessura das patas dos animais foram avaliadas.

4.4 Isolado e cultivo de Paracoccidioides brasiliensis

Paracoccidioides brasiliensis (Pb) isolado virulento Pb18 foi mantido e

adaptado para a fase leveduriforme no meio Sabouraud-Dextrose (Sanofi, França) e

mantido a 37 oC. 5 a 7 dias antes da inoculação, foi transferido para o meio líquido

Mc Veigh & Morton (McVM), quimicamente definido, a 35 °C (CASTAÑEDA et al.,

1988; HANDAM et al., 1998; RESTREPO; ARANGO 1980; SINGER-VERMES et al.,

1992).

4.5 Infecção e Terapia

4.5.1 Preparo do inóculo

O fungo foi coletado e lavado 3 vezes com tampão fosfato (PBS pH 7,2).

Após a última lavagem, a solução permaneceu imóvel, por alguns minutos, para que

ocorra a sedimentação dos grumos celulares. A suspensão, contendo células

individuais ou com poucos brotamentos, foi coletada e a contagem realizada em

câmara de Neubauer.

4.5.2 Determinação da viabilidade das leveduras

A viabilidade das leveduras foi determinada através do corante azul de

Tripan (Sigma, St. Louis, EUA). Todos os procedimentos cirúrgicos executados no


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experimento foram realizados com suspensão fúngica com viabilidade entre 90 e

95%.

4.5.3 Infecção Intratraqueal (i.t.)

Os camundongos foram infectados por via intratraqueal conforme descrito

previamente por Taborda et al. (1998). Foi utilizado inóculo contendo 3x105

leveduras em 50 μl para cada camundongo. O procedimento foi realizado com os

animais sob anestesia (80 mg/kg de ketamina e 10 mg/kg de xilazina).

Quando os animais se apresentaram insensíveis à dor (após dez minutos), foi

feita uma pequena incisão longitudinal na pele do pescoço e a traquéia ficou

exposta. Após a inoculação do fungo, a incisão foi suturada e os animais colocados

sob uma fonte moderada de calor para controlar a hipotermia transitória produzida

pela anestesia, até acordarem.

4.5.4 Terapia com agentes antimicrobianos

As doses foram definidas com as concentrações de: 15 – 3 mg kg-1 de peso

para sulfametoxazol-trimetoprim (Bac-Sulfitrin® Ducto, Brasil) e 10 mg kg-1 de peso

para o Itraconazol (Janssen-Cilag®, Brasil). Os antimicrobianos foram administrados

pela via intraperitoneal, todas as quantidades administradas das drogas foram

baseadas nos tratamentos seguidos em humanos. As drogas foram conservadas e

mantidas em temperatura ambiente e preparadas com uma prévia dissolução em 2%

de DMSO. Para o sulfametoxazol/trimetoprim (Bac-sulfitrin, Ducto, Brasil), a diluição

foi feita em PBS, com 2% de DMSO.

4.5.4.1 Primeiro protocolo

A terapia foi iniciada 15 dias após a infecção intratraqueal e realizada por um

período de 30 dias consecutivos, com administração, a cada 24 h, para Itraconazol e

para sulfametoxazol/Trimetoprim, com as concentrações acima citadas, após 15 dias

de infecção todos os antimicrobianos foram administrados por via intraperitonial. O


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peptídeo P10 foi aplicado em 4 doses 1 por semana. Os animais foram sacrificados

após 45 dias de infecção.

4.5.4.2 Segundo protocolo

A terapia foi iniciada 30 dias após a infecção intratraqueal e realizada por um

período de 30 dias consecutivos, com administração, a cada 24 h, para Itraconazol e

para sulfametoxazol/Trimetoprim, com as concentrações acima citadas, após 30 dias

de infecção todos os antimicrobianos foram administrados por via intraperitonial. O

peptídeo P10 foi aplicado em 4 doses 1 por semana. Os animais foram sacrificados

com 60 dias de infecção.

4.6 Síntese e Purificação do Peptídeo (P10)

O peptídeo foi sintetizado e purificado no departamento de Biofísica, da

UNIFESP, assim como descrito por Taborda et al. (1998). Os estudos de

biodistribuição foram realizados com o Peptídeo P10 fabricado pela Peptide 2.0

(Peptide 2.0., Inc. Chantilly, Virginia, VA., USA), com uma pureza de 98%,

determinada por análise de HPLC e MS.

4.7 Imunização dos camundongos

Camundongos BALB/c machos pesando aproximadamente 25 g foram

imunizados subcutaneamente com 20 μg do peptídeo 10 (P10) em adjuvante

completo de Freund na primeira dose; as subseqüentes (3 doses; 1 por semana)

foram administradas com adjuvante incompleto. A emulsão (50 µL) foi injetada em

cada animal em uma das patas traseiras na primeira dose e as últimas três foram

por via intraperitoneal. Os camundongos controles receberam somente o adjuvante

sem o peptídeo.


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4.8 Grupos utilizados

Foram utilizados 10 grupos de camundongos machos BALB/c, contendo 6

animais cada (os mesmos grupos de animais foram utilizados no desenvolvimento

dos dois protocolos), sendo:

1) Sham NI: não infectado, não imunossuprimido e não tratado; 2) infectado

NI: infectado com Pb18, não imunossuprimido e tratado com PBS; 3) Sham:

Imunossuprimido, não infectado; 4) infectado: infectado com Pb18, imunossuprimido

e tratado só com PBS; 5) Adj: infectado, imunossuprimido e imunizado somente com

adjuvante completo e/ou incompleto de Freud; 6) ITRA: infectados,

imunossuprimidos e tratados somente com itraconazol, 7) SULFA: infectados,

imunossuprimidos e tratados somente com sulfametoxazol/trimetoprim; 8) P10:

infectado, imunossuprimido e imunizado somente com P10; 9) Itra+P10: infectado,

imunossuprimido tratado com sulfametoxazol/trimetoprim e imunizado com P10; 10)

Sulfa+P10: infectado, imunossuprimido tratado com sulfametoxazol/trimetoprim e

imunizado com P10.

4.9 Análise da carga fúngica através das unidades formadoras de colônias

O grau de infecção foi caracterizado em animais tratados com drogas em

associação, ou não, com a imunização com P10 e seus respectivos controles,

através da recuperação de fungos viáveis do pulmão, fígado e baço dos

camundongos, após 45 e 60 dias de infecção, em meio Brain Heart Infusion (BHI)

(Difco, laboratories, Detroit, MI, USA), suplementado com 4% (vol/vol) de soro

bovino (Gibco, NY, USA) e 5 % de filtrado de cultura do isolado 192 do P.

brasiliensis, streptomicina/penicillina 10 IU ml-1 (Cultilab, Brasil) e cicloheximida 500

mg ml-1 (Sigma, St Louis, Mo). Após a morte dos animais com punção cardíaca, os

órgãos (pulmão, baço e fígado) foram extirpados, divididos ao meio e pesados,

imediatamente. Com o auxílio de um homogeneizador manual, as células foram

rompidas em 1 ml de PBS; dessa solução, 100 L foram plaqueados e incubados a

37 °C, e as colônias foram contadas, com 10 dias de incubação.


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4.10 Dosagem de citocinas do homogeneizado de pulmão pelo método de CBA

A quantificação das citocinas foi feita pelo método de Cytometric Bead Array

(CBA) (FACSCalibur, EUA) no software BD FACSCompTM & BD CaliBRITETM

Beads. Antes de analisar as amostras, foi realizada a curva padrão com as diluições

(1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256) definindo a concentração do padrão

entre 20 - 5000 pg/ml. Após a realização do padrão, foi feita a reconstituição do Mix,

no qual são adicionados 10µl do anticorpo específico para cada citocina em cada

amostra. Todos os procedimentos foram realizados a temperatura de 4 ºC. Após

adicionar o mix foram acrescentados 50µl do anticorpo de detecção fluorocromo, PE,

por amostra e estas foram protegidas da luz por 2 horas. Foram preparadas as

Setap Beads, marcando três tubos (A, B e C): nos tubos B e C foram adicionados

50µl dos reagentes de marcação, FITC e PE respectivamente, e foram adicionados

400µl da solução de lavagem. No tubo A foram adicionados 450 µl desta solução. Os

Setap Beads foram incubados a temperatura ambiente por 30 minutos, protegidos

da luz. Após este procedimento, as amostras foram analisadas, calibrando-se o

citômetro com o CaliBRITE Beads e os resultados foram obtidos em pg/g de tecido.

4.11 Detecção por ELISA de anticorpos (IgG total, IgG2a, IgG2b e IgG1) no soro

de camundongos BALB/c anérgicos infectados i.t com P. brasiliensis e

imunizados ou não com o peptídeo P10. (2° protocolo)

Foram utilizadas placas de microtitulação de 96 poços, que foram

sensibilizadas com 200 mg de gp43, durante 12 h, a 4-8 ºC. As placas foram, então,

lavadas com PBS Tween 20 0,05% (PBS-T) e bloqueadas com BSA 1%, a 37 ºC.

Após nova lavagem, foram adicionados 50 µl de soro de camundongos, diluídos,

inicialmente, a 1/200, seguindo-se titulações com diluições subseqüentes na razão

2, e incubação, por 1 hora, a 37 ºC. Um anticorpo específico para cada isotipo

marcado com biotina (IgG1, IgG2a, IgG2b, IgG3, IgM e IgG total – BD PharMingen,

San Diego) foi adicionado após as lavagens, seguindo-se incubação, por 1hora, a 37

ºC. As placas foram novamente lavadas com PBS-T e os conjugados enzimáticos

adicionados (streptoavidina-fosfatase alcalina). A reação foi desenvolvida pela


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adição de substrato para fosfatase (Sigma, St. Louis). A leitura foi realizada em

microleitor de ELISA (Labsystems iEMS Analyser, Helsinki), com filtro de 405 nm.

As amostras foram consideradas positivas para anticorpos anti-gp43, quando

a D.O. (405 nm) fosse superior a 0,200, segundo o “cut off” obtido pela média das

absorbâncias obtidas para os animais-controle, durante a realização dos testes.

4.12 Dosagem de óxido nítrico (NO)

O sobrenadante de cultura celular e o homogeneizado do tecido pulmonar

foram utilizados para determinar a produção de óxido nítrico, esta dosagem foi

realizada no analizador de quimioluminescência (NOATM280, Sievers Inc., USA). A

curva de calibração foi determinada usando padrões de nitrato de sódio. Usando o

analizador NOATM280, o nitrato foi reduzido a óxido nítrico (NO) com Vanadium (III)

a 90 ºC, e o NO formado foi detectado por fase gasosa de quimioluminescência após

reação com o ozônio.

4.13 Análise imunohistoquímica através da reação de estreptovidina-biotina-

peroxidase

Os fragmentos de tecido das amostras foram gradativamente congelados em

nitrogênio líquido e armazenados no freezer -80ºC até análise. As secções

congeladas foram processadas no criostato (Leica CM1850) e os cortes de 5

micrômetros foram colocados em lâminas de poli-L-lisina (Sigma-Aldrich) e fixadas

com acetona para a realização da análise imunohistoquímica (CAVASSANI et al.,

2006).

No momento da análise por imunohistoquímica, as lâminas foram incubadas

com tampão de lavagem PBS (pH 7,6) por 3 minutos, após este período foi feito o

bloqueio de peroxidase endógena com solução 3% de peróxido de hidrogênio (30% -

Merck, Germany) em Metanol (Synth, Brasil) sob agitação, por 5 minutos, após o

bloqueio os cortes foram lavados com tampão de lavagem (2 vezes de 3 minutos

cada). Em seguida, foi aplicado sobre os cortes BSA 2% para o bloqueio de reações

inespecíficas, por 2 minutos. A solução bloqueadora foi removida com tampão de

lavagem (3 vezes de 3 minutos cada). Além disso, foi empregada uma solução


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tampão TBS (Tris-buffered salina – salina tamponada com Tris) (Pierce Inc.) sobre

os cortes de tecido para o bloqueio da biotina endógena. Às lâminas foram

adicionados os anticorpos primários específicos para células CD11b+, Ly-6G/Ly-6C+

e L3T4+ nas diluições adequadas em PBS/BSA 1%, posteriormente incubadas

“overnight” a 4 °C. No caso do anticorpo anti-L3T4, a incubação foi de 30 minutos a

4 °C. O anticorpo primário foi lavado com tampão de lavagem (3 vezes de 3 minutos

cada). Posteriormente, as lâminas foram incubadas com o anticorpo secundário

biotinilado (Vector Laboratories.), diluído 1:500 em PBS, por 30 minutos à

temperatura ambiente. O anticorpo secundário foi lavado com tampão de lavagem (3

vezes de 3 minutos cada). A seguir, as lâminas foram incubadas com estreptovidina-

peroxidase (1 mg mL-1, vector laboratories) na diluição 1:100 em PBS por 30 minutos

à temperatura ambiente e a reação foi revelada com tetra-hidrocloreto de 3,3

diaminobenzidina (Sigma Biochemical Co.). As reações foram interrompidas após a

visualização de cor marrom nos cortes de tecidos e a seguir, as lâminas foram

contracoradas com hematoxilina de Mayer, desidratadas e montadas em solução

Permont e observadas em microscópio óptico (Nikon) com aumentos de 40x, 100x,

250x e 400x.

Lâminas com cortes de tecido de camundongos BALB/c imunossuprimidos e

infetados foram utilizadas como controles, lâminas com estes cortes e com ausência

do anticorpo primário foram empregadas para avaliar a especificidade da reação.

4.14 Ensaio de proliferação celular

Após uma semana da última imunização ou 60 dias após a infecção, os animais

foram sacrificados, retirando-se o baço para pesquisa de células T reguladoras e

células T de memória. As células deste tecido foram desagregadas através de

maceração em meio RPMI estéril e as hemácias foram lisadas. As células foram

contadas em Câmara de Neubauer e a viabilidade destas foi determinada utilizando-

se o teste de exclusão com azul de Trypan, com a viabilidade em torno de 90%. As

células do baço foram plaqueadas em placas de 96 poços, fundo chato, na

concentração de 4x105 células/poço, sendo que em cada poço o volume final foi de

200 μl. O meio utilizado foi RPMI 1640 suplementado com 20 mM NaHCO3, 10 mM

Hepes, 100 U/ml de penicilina, 100 mg/ml de estreptomicina, 2 mM de L-glutamina,


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50 μM de β-mercaptoetanol, 5 mM de piruvato de sódio, 100 mM de aminoácidos

não essenciais e 10% de soro fetal bovino. Às células dos poços controle

acrescentou-se Concanavalina A (1000 ng/poço) e o peptídeo P10 (20 μg/ml) a

todos os outros poços. As placas foram mantidas por 144 horas em estufa com 5%

de CO2 a 37 ºC, retirando-se a Concanavalina A após 48 horas. Para análise da

proliferação celular foi utilizado o sal de tetrazolium MTT (Thiazolyl Blue Tetrazolium

Bromide – Sigma, St. Louis) conforme protocolo adaptado de Mosmann (1983).

Após as 144 horas, foi acrescentado 50 μl/poço de MTT (1 mg/ml) e as placas

retornaram a estufa com 5% de CO2 a 37 ºC por quatro horas. Para parar a reação e

dissolver os cristais de formazana formados, adicionou-se 100 μl/poço de

Isopropanol – HCl 0,04N. Procedeu-se à leitura em leitor de ELISA no comprimento

de onda de 590 nm.

4.15 Dosagem de citocinas do sobrenadante de cultura celular pelo método de

ELISA

Procedeu-se a dosagem das seguintes citocinas quantitativamente pelo

método de ELISA de captura (BD Pharmingen, San Diego): IFN-, IL-12, IL-4 e IL-10.

Placas para ELISA foram sensibilizadas com 100 l dos anticorpos de captura e

mantidas a 4 ºC overnight. Após, procedeu-se a lavagem dos poços com PBS-

Tween 20 0,05% e bloqueadas com 200 l de Assay Diluent (PBS-10% Soro Fetal

Bovino) por uma hora, a temperatura ambiente. Após serem novamente lavadas,

acrescentou-se aos poços 100 l das amostras (sobrenadante de cultura celular) ou

das citocinas recombinantes de camundongos (padrão), realizando-se as diluições

necessárias e as placas foram incubadas a temperatura ambiente por duas horas.

Após esse período, as placas foram novamente lavadas com PBS – Tween 20

0,05% e foram adicionados 100 l do conjugado, composto dos anticorpos de

detecção e a enzima. Assim, as placas foram novamente incubadas por uma hora, à

temperatura ambiente. Após, procedeu-se a mais uma etapa de lavagem dos poços

e acrescentou-se 100 l do substrato da enzima (TMB substrate reagent – BD

Pharmingen, San Diego). As placas foram incubadas no escuro, a temperatura

ambiente por trinta minutos e a reação foi parada com 50 l/poço de H2SO4 2N. A


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leitura foi realizada em leitor de ELISA no comprimento de onda de 450 nm

(Multskan EX, USA).

4.16 Imunofenotipagem por citometria de fluxo

Após o estímulo com o peptídeo P10 por 144 horas, as células do baço foram

retiradas das placas e centrifugadas a 450 x g por cinco minutos a 4 ºC. O

sobrenadante foi coletado e armazenado a -70 ºC para posterior análise de

citocinas. Às células foi adicionado 80μl de sobrenadante de cultura do hibridoma

24G2 contendo anticorpos anti-CD16/CD32 (bloqueador de fração Fc) durante trinta

minutos a 4 ºC. Posteriormente, as células foram marcadas para as moléculas de

superfície, utilizando-se os seguintes anticorpos, anti-CD4-FITC (clone RM4-5, 0,5

μg/106 células), anti-CD8-PerCP (clone 53-6.7, 0,5 μg/106 células), anti-CD44-PE

(clone IM7, 0,5 μg/106 células) e anti-CD25-PE (clone PC61, 0,5 μg/106 células) e

incubadas por trinta minutos a 4 ºC. Após este período, as células foram lavadas

com PBS acrescido de 3% de Soro Fetal Bovino e, a seguir, realizou-se a fixação e

permeabilização das células para então proceder a marcação para Foxp3 utilizando

o anticorpo anti-Foxp3-PE (clone FJK-16s, 0,5 μg/106 células). A fixação e

permeabilização foi realizada utilizando o kit “Foxp3 Staining Buffer Set” (e-

Bioscience), seguindo as instruções do fabricante. Como controle foram utilizadas

células não marcadas e tubos FMO (fluorescence minus one), os quais apresentam

marcação para todas as moléculas de interesse menos uma delas. As células foram

incubadas novamente por trinta minutos a 4ºC e lavadas com PBS acrescido de 3%

de Soro Fetal Bovino. Após a lavagem, foram adicionados 300 μl de PBS acrescido

de 3% de Soro Fetal Bovino e procedeu-se a aquisição dos dados em citômetro de

fluxo (FACScalibur). A análise das amostras adquiridas foi realizada utilizando o

software FlowJo 7.2.4 (TreeStar).

4.17 Contagem diferencial dos Leucócitos

Lâminas de vidro foram utilizadas para realizar os esfregaços de uma gota de

sangue, extraída do plexo ocular dos animais imunizados ou não com o peptídeo

P10. Estes esfregaços foram tratados com o kit Panótico Rápido (Laborclin Brasil


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Ltda). Para evitar erros de contagem, esta foi feita percorrendo a lâmina toda porém,

descrevendo um zigue-zague. Aconselha-se iniciar a contagem da região média do

esfregaço (corpo) para cauda. Foi realizada a contagem de 100 leucócitos para

obter o resultado em porcentagem.

4.18 Curva de sobrevida

Cada grupo com 5 animais foram infectados com 3x105 leveduras de Pb18.

Como controles foram utilizados animais anérgicos infectados e tratados com PBS.

A mortalidade foi registrada diariamente por um período de 200 dias e os

camundongos foram mantidos em condições livre de patógenos no biotério de

experimentação animal do Departamento de Microbiologia do Instituto de Ciências

Biomédicas da Universidade de São Paulo.

4.18.1 Curva de sobrevida de camundongos knockout iNOS-/-

Com o propósito de determinar o papel do óxido nítrico, no processo de

imunização com o peptídeo P10, camundongos imunossuprimidos, knockout para

iNOS, foram infectados e imunizados ou não com o peptídeo P10. A sobrevida

destes animais foi observada a por um período de 200 dias, as condições de

manutenção foram idênticas à curva de sobrevida realizada nos camundongos

BALB/c anérgicos.

4.19 Histopatologia

Uma fração do pulmão foi acondicionada em um tubo contendo formalina

(Merck, Alemanha) 10% e enviada para realização da análise histopatológica no

laboratório de Histologia do Departamento de Imunologia da Universidade de São

Paulo. Os cortes foram submetidos à coloração HE e Gomori-Grocott.


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4.20 Determinação da fibrose pulmonar

Pulmões de camundongos BALB/c imunossuprimidos e infectados com Pb18,

foram retirados, fixados em formalina tamponada 10 % e enviada para realização da

análise histopatológica no laboratório de Histologia do Departamento de Imunologia

da Universidade de São Paulo. Os cortes foram submetidos à coloração Gomori

Silver-reticulina, para confirmar as mudanças encontradas na organização das fibras

de reticulina (colágeno tipo III), e a coloração tricrômico de Mason foi usada para

identificar as fibras de colágeno do tipo I nos pulmões.

4.21 Marcação com radiofármaco

Uma molécula modificada do peptídeo sintético P10, na qual a cadeia de 15

aminoácidos (QTLIAIHTLAIRYAN) associada ao 6-hidrazino nicotinamida (HYNIC),

foi utilizada para avaliar a biodistribuição do peptídeo no modelo murino. O HYNIC-

P10 foi sintetizado pela PEPTIDE 2.0 (Chantilly, VA, United States). A conjugação

do HYNIC-P10 foi realizada com 95% de grau de pureza, analisada por

cromatografia líquida ligada à espectrometria de massa.

O conjugado HYNIC-P10 foi marcado com o radioisótopo tecnécio-99m. A

uma alíquota de 10 µg do HYNIC-P10 foram adicionados a 40 mg de tricina e 2 mg

de acido nicotínico, dissolvidos em 0,5 mL de tampão fosfato 0,1 M (pH 7,2)

previamente nitrogenado. Em seguida, 10 µg de cloreto de estanho bihidratado

dissolvido em acido clorídrico (HCl) 0,1 N, também nitrogenado, foi adicionado,

seguida da solução (0,5 mL) de pertecnetato de sódio (Na99mTcO4). A reação

ocorreu por aquecimento a 100 ºC por 20 min (FAINTUCH et al., 2005 ).

O produto HYNIC-P10-99mTc (0,1 mL) com atividade aproximada de 74 MBq

(2 mCi) foi administrado aos animais pela via intravenosa (veia caudal). Os animais

foram sacrificados por deslocamento cervical aos 5, 30, 60, 120, 240 e 360 minutos

após a injeção do radioproduto.

Para os estudos de biodistribuição do P10 foram utilizados camundongos da

linhagem BALB/c com 6 a 8 semanas de vida e pesando de 25 a 30 gramas, os

quais foram fornecidos pelo biotério do Instituto de Pesquisas Energéticas e

Nucleares (IPEN/CNEN, SP).


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Os órgãos (rins, baço, fígado, pulmões, cérebro, coração, intestino grosso e

delgado, pâncreas, estômago, músculos, ossos, cauda) foram dissecados, pesados

e transferidos para tubos de contagem radioativa. A contagem foi realizada em

contador de radiação gama tipo poço de NaI(Tl), utilizando-se como padrão a

mesma dose injetada nos animais. Para o cálculode captação por órgãos e tecidos,

descontou-se a radioatividade da cauda (por ser o local de administração do material

radioativo), do padrão.

O cálculo da captação do radioproduto por órgão foi realizado pela equação:

% DI = (CPM órgão / CPM padrão) X 100

Em que %DI = Porcentagem da dose injetada e CPM = Contagem por minuto.

4.22 Estudos de imagem por biomarcação

A aquisição de imagem cintilográfica planar de animais foi efetuada em gama-

câmara equipada com um colimador de baixa energia e alta resolução, ângulo de

90º e uma matriz de 256 x 256 X 16, durante 180 segundos. O estudo foi realizado

em animais previamente anestesiados aos 30, 60 e 120 min após administração dos

radiotraçadores. A captação em regiões de interesse (ROI) foi quantificada

considerando-se uma atividade do animal equivalente a 100% (FAINTUCH et al.,

2008).

4.23 Determinação da atividade citotóxica in vivo do peptídeo P10

O ensaio de toxicidade foi realizado de duas formas, em tecidos animais,

onde camundongos BALB/c com idade entre 6 – 8 semanas foram imunizados com

100 ug do peptídeo P10 pela via i.p, 3 vezes a cada 24 h, na ausência de adjuvante.

No quinto dia após o começo do tratamento, os animais foram sacrificados por

dislocamento cervical e os órgãos (pulmão, baço, rins e fígado) foram dissecados. A

histologia dos órgãos foi realizada por coloração H&E.


Muñoz J. E

54

4.24 Determinação da atividade citotóxica in vitro do peptídeo P10

Para os ensaios de citotoxicidade do peptídeo P10, células das linhagens,

endotelial (Human Umbilical Vein Endothelial Cells) HUVEC (5 x 103) e fibroblasto

murino MEFs (mouse embryonic fibroblasts), (1 x 104) foram distribuidas em placas

de 96 poços. Após 24 h, o peptídeo foi acrescentado às células em diferentes

concentrações (100, 80, 60, 40 e 20 µg/mL) e as mesmas foram incubadas a 37 ºC e

5% CO2 por 20 horas. Para determinação da viabilidade celular após este período,

as células foram coletadas com Tripsina e contadas em câmera de Neubauer,

utilizando o teste de exclusão de Trypam Blue (ARRUDA et al., 2012). Estes

experimentos de viabilidade celular foram realizados em triplicata e os resultados

expressos em número total de células por poço.

4.25 Análise estatística

Análise Estatística. Os resultados foram analisados utilizando-se o software

GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA) e a análise de variância

(ANOVA) foi realizada seguida do pós-teste de Tukey. O resultado foi considerado

significativo quando o p< 0,05. Para os dados obtidos do experimento de

sobrevivência foi utilizado o teste Long-Rank (Matel-Cox).

Resultados

Muñoz J. E

55

5 RESULTADOS

5.1 Padronizações do modelo de anergia com o corticóide sintético “fosfato de

dexametasona” em camundongos BALB/c

Ao examinar o efeito da imunossupressão com dexametasona foi observada

uma nítida redução no número global de leucócitos no sangue periférico, de

camundongos tratados com dexametasona, a contagem dos leucócitos apresentou

redução progressiva no decorrer do tempo no experimento (Figura 1). O grupo

controle não recebeu o fosfato de dexametasona na água de beber, e manteve no

décimo, vigésimo e trigésimo dia a média na contagem de leucócitos desde o início

dos experimentos.

Figura 1 - Contagem do número de leucócitos no sangue periférico.

Contagem de Leucócitos

Contr

ole 10

20

30

0

1000

2000

3000

4000

***

****

Dias de Tratamento

# d

e L

eu

co

cit

os

po

r m

m3

A contagem foi realizada (leucócitos/mm3) no 10°, 20° e 30° dia de protocolo, nos camundongos tratados com dexametasona em dose imunossupressora 0,15 mg/kg-

1. **

p<0,01 *** p<0,0001 diferença entre animais controle (não tratados com dexametasona) e animais tratados com dexametasona.

Outro método para observar o efeito imunossupressor da dexametasona foi

realizado, com o propósito de identificar o tempo necessário para completar a

anergia dos animais. A reação de HTT ou teste de hipersensibilidade tardia foi feito

Resultados

Muñoz J. E

56

em camundongos BALB/c, infectados intratraquealmente com 3x105 leveduras de

Pb18, na qual foi observada uma diminuição significativa na inflamação apresentada

na extremidade posterior dos animais a partir do décimo quinto dia, mas a partir do

vigésimo dia podemos considerar os camundongos anérgicos, já que não

respondem mais ao teste de HTT, conforme o observado na figura 2 (APÊNDICE C).

Figura 2 - HTT realizado em camundongos BALB/c.

O HTT foi realizado em camundongos BALB/c infectados i.t (○) e camundongos BALB/c infectados i.t e imunossuprimidos com 0,15 mg/kg-1 de dexametasona (●). As barras de erro indicam o desvio padrão. * p<0,05. Fonte: Marques et al., 2008.

O estado de anergia foi observado depois do dia vigésimo dia de tratamento

com dexametasona na água de beber, uma condição que causa a morte de 100%

dos camundongos infectados entre os dias 70 e 80 de infecção experimental (Figura

3) (APÊNDICE A, Artigo 2).

É importante resaltar que o fosfato de dexametasona provocou uma drástica

redução de peso (relativo e absoluto) nos animais que receberam o tratamento com

este corticoide. Já ao observar os órgãos destes animais se destacou a diminuição

do baço (Figura 4).

Anérgicos

Dias de Infecção

Resultados

Muñoz J. E

57

Figura 3 - Sobrevida de camundongos BALB/c infectados i.t com 3x105 células

leveduriformes de P. brasiliensis e tratados com dexametasona.

Animais tratados com dexametasona (0.15 mg/kg) na água de beber (●) grupo controle que não recebeu tratamento com Dexametasona (○). Fonte: Marques et al., 2008.

Anérgicos Controle

Dias de Infecção

Resultados

Muñoz J. E

58

Figura 4 - Baços de camundongos BALB/c submetidos ao tratamento com

Dexametasona.

Baços de animais submetidos ao tratamento com fosfato de dexametasona (0,15 mg/kg) por 20 dias (A) e de animais controle não tratados com o fosfato de dexametasona (B). Estes baços são de animais da mesma idade e não infectados.

Com a padronização do modelo de anergia em camundongos BALB/c

infectados intratraquealmente com P. brasiliensis estabelecido, conforme

apresentado acima; nos experimentos seguintes avaliamos o efeito aditivo do

peptídeo 10 (P10) no modelo experimental, com objetivo de mimetizar o estado

anérgico, comum em pacientes com as formas aguda e subaguda da doença.

5.2 Análise da carga Fúngica através das Unidades Formadoras de Colônias

1° protocolo: (Os animais foram sacrificados com 45 dias de infecção)

Neste protocolo, os animais foram infectados intratraquealmente com 3x105

células leveduriformes de P. brasiliensis, e a terapia iniciou-se após 15 dias da

infecção, com doses administradas, intraperitonealmente, de 10mg/kg-1 de peso para

o itraconazol e 15 – 3/mg kg-1 para sulfametoxazol-trimetoprim, durante 30 dias,

diariamente. O sacrifício foi após 45 dias de infecção.

Ao sacrificar os animais, a simples vista, observamos que os pulmões dos

animais controle infectados e tratados com o fosfato de Dexametasona

apresentaram um aumento no tamanho. O estado destes órgãos estava bastante

comprometido pelo fungo e nos outros órgãos, também foi observada uma

Resultados

Muñoz J. E

59

agressividade maior da infecção. Estes animais apresentaram um aumento nas

UFCs quando comparados aos animais que não receberam a Dexametasona. Já

nos animais infectados que receberam o tratamento quimioterápico com os

antifúngicos, houve uma redução significativa da carga fúngica, igual aos que foram

somente imunizados com o peptídeo P10.

Os melhores resultados foram obtidos ao associar o tratamento

quimioterápico à imunização com o P10, onde foi observada uma queda significativa

da carga fúngica. A diminuição da carga fúngica foi similar tanto no grupo tratado

com Itraconazol, associado ao P10, como no grupo de animais tratado com

Sulfametoxazol/Trimetoprim, associado ao P10. Estes grupos de animais também

mostraram uma diminuição significativa da disseminação das leveduras para outros

órgãos como fígado e baço (Figura 5).

É importante ressaltar que animais imunossuprimidos e infectados com P.

brasiliensis com 30 dias o mais de infecção apresentam perda de peço e alterações

na pelagem. Nos grupos de animais que receberam o tratamento quimioterápico,

associado à imunização com o P10, não foram observadas estas alterações

fenotípicas, o que evidencia a eficácia do tratamento (dados não mostrados).

Resultados

Muñoz J. E

60

Figura 5 - UFCs de pulmão (P), baço (B) e fígado (F) de camundongos BALB/c

anérgicos, com 45 dias de infeção.

Animais Anérgicos

P B F P B F P B F P B F P B F P B F P B F P B F

0

5000

10000

15000

45 Dias

Infectado Infectado Adj. P10 Itra. Sulfa. Itra+P10 Sulfa+P10

***

***

***

UF

C/g

de t

ecid

o

Tratados com Dexametasona

Os animais foram infectados intratraquealmente e tratados (15 dias após i.t.) com itraconazol e sulfametoxazol-trimetoprim, por 30 dias, associadas, ou não, à imunização com P10, e sacrificados após 45 dias de infecção. Foram utilizados, como controles, animais somente infectados e não tratados/imunizados (controle),* p<0,05 ** p<0,01 *** p<0,0001 quando comparado com os animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste Tukey.

5.3 Análise da carga Fúngica através das Unidades Formadoras de Colônias

2° protocolo: (Os animais foram sacrificados com 60 dias de infecção)

Neste protocolo, os animais foram infectados intratraquealmente com 3x105

células leveduriformes de P. brasiliensis, e a terapia iniciou-se após 30 dias da

infecção, com doses administradas, intraperitonealmente, de 10 mg/kg-1 de peso

para o itraconazol e 15 – 3/mg kg-1 para sulfametoxazol-trimetoprim, durante 30 dias,

diariamente. O sacrifício foi, portanto, após 60 dias de infecção.

Os grupos de animais tratados apenas com itraconazol e sulfametoxazol-

trimetoprim, não apresentaram diminuição significativa das UFCs e a disseminação

Resultados

Muñoz J. E

61

da doença foi maior do que nos animais tratados com itraconazol e sulfametoxazol-

trimetoprim, associado à imunização com P10, onde observamos uma queda

significativa na carga fungica nos pulmões, quando comparados com os pulmões do

grupo controle e também não houve disseminação da doença a outros órgãos, como

o baço e o fígado (Figura 6). Mostrando assim, uma eficácia no controle da

paracoccidioidomicose experimental.

Figura 6 - UFCs de pulmão (P), baço (B) e fígado (F) de camundongos BALB/c anérgicos, com 60 dias de infecção.

P B F P B F P B F P B F P B F P B F P B F P B F

0

5000

10000

15000

20000

25000

Infectado Infectado Adj. P10 Itra. Sulfa. Itra.+P10 Sulfa.+P10

60 Dias

*****

*


UF

C/g

de t

ecid

o

Os animais foram infectados intratraquealmente e tratados (30 dias após i.t.) com itraconazol e sulfametoxazol-trimetoprim, por 30 dias, associado, ou não, à imunização com P10, e sacrificados após 60 dias de infecção. Foram utilizados, como controles, animais somente infectados e não tratados/imunizados (controle). * p<0,05 ** p<0,01 *** p<0,0001 quando comparado com os animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste Tukey.

Com a intenção de definir o papel do P10 como um adjuvante imunológico no

tratamento da PCM experimental, associado às drogas, animais infectados foram

analisados em dois protocolos diferentes. Nos dois procedimentos, realizados de

quimioterapia, observamos melhora significativa nos animais infectados com P.

brasiliensis e tratados com antifúngicos e imunizados com P10.

Resultados

Muñoz J. E

62

5.4 Análise das citocinas IFN-, IL-12, TNF-α, IL-10, IL-6, IL-8, MCP-1, IL-4 e IL-

2 nos camundongos BALB/c anérgicos infectados e tratados com drogas

antifúngicas, associadas, ou não, à imunização com P10

Uma fração dos pulmões dos camundongos BALB/c anérgicos, infectados e

tratados com as drogas em associação, ou não, com o P10, foi pesado e

homogeneizado, na presença de inibidores de protease. O sobrenadante foi

conservado a -80 oC, até o momento do ensaio. A quantificação das citocinas foi

feita através do programa Cytometric Bead Array (CBA) (FACSCalibur, EUA).

Ao observar e comparar os resultados obtidos nos dois protocolos (após 45 e

60 dias de infecção) não foi possível identificar diferença significativa entre os

protocolos, na secreção de citocinas dos perfis tanto Th1 como Th2.

É importante ressaltar que ocorreu um aumento significativo nos níveis de

citocinas pró-inflamatórias, como IFN-, IL-12, TNF-α e IL-2 e diminuição das

citocinas IL-4 e IL-10 (citocinas imunoreguladoras), nos grupos que receberam o

tratamento com itraconazol e sulfametoxazol-trimetoprim associados ao P10. Estas

análises foram comparadas ao grupo controle (animais tratados com dexametasona

e infectados), os quais apresentavam uma fisiologia aproxima do perfil da resposta

da PCM aguda humana; foco do trabalho em questão.

A figura 7 indica os valores de IFN-, onde podemos observar que os

imunizados com P10 tiveram um aumento significativo desta citocina, tanto após 45

como 60 dias de infecção, enquanto que os tratados somente com as drogas

apresentaram níveis mais baixos, em relação ao grupo infectado e tratado com

dexametasona.

Os animais que receberam somente o adjuvante não apresentaram diferença

significativa. Entretanto, nos animais que receberam dexametasona foi observada

uma diminuição desta citocina quando comparado com os animais infectados e

sham que não receberam o tratamento com o corticóide.

Resultados

Muñoz J. E

63

γ

Figura 7 - Dosagem da citocina IFN-γ, pelo método Cytometric Bead Array (CBA)

(FACSCalibur, EUA), de homogeneizado dos pulmões de camundongos BALB/c anérgicos, infectados por via intratraqueal com 3x105 células de P. brasiliensis e tratados com as drogas antifúngicas, associadas, ou não, à imunização com P10, e sacrificados com 45 e 60 dias de infecção.

IFN-

0

20

40

60

80

ShamInfectado Infectado P10 Adj. Itra. Itra.+P10Sulfa. Sulfa.+P10Sham

60 dias 45 dias

Tratados com Fosfato de Dexametasona

***

***

*

*****

pg

/ml

* p<0,05 ** p<0,01 *** p<0,0001 quando comparado com os animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste

Tukey.

Outra citocina pro-inflamatória que desempenha um papel importante

direcionando a uma resposta imune do tipo Th1 é a IL-12. Como podemos observar

na figura 8 houve um aumento significativo nos níveis de IL-12, no grupo de animais

imunizados com P10 e também nos camundongos tratados com itraconazol e

sulfametoxazol-trimetoprim em associação ao P10, quando comparados ao grupo de

animais infectados e tratados com dexametasona. Esse resultado pode indicar um

perfil de resposta polarizado para o tipo Th1, fundamental como resposta protetora à

infecção pelo fungo.

Os grupos de animais que receberam somente as drogas e o adjuvante de

Freund não apresentaram aumento significativo na secreção de IL-12.

Resultados

Muñoz J. E

64

Figura 8 - Dosagem da citocina IL-12 pelo método Cytometric Bead Array (CBA)


IL-12

0

100

200

300

ShamInfectado Infectado P10 Adj. Itra. Itra.+P10Sulfa. Sulfa.+P10Sham

Tratados com Fosfato de Dexametasona

60 dias 45 dias

***

*

**

*

pg

/ml

* p<0,05 ** p<0,01 quando comparado com os animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste Tukey.

Na figura 9, a qual indica os valores de TNF-α, é possível observar que o

grupo de animais imunizados somente com P10 e, os grupos imunizados com o

P10, associado ao tratamento com itraconazol e sulfametoxazol-trimetoprim

apresentaram um aumento significativo desta citocina no hogeneizado dos pulmões,

após 45 e 60 dias de infecção. Enquanto que os animais tratados somente com as

drogas apresentaram níveis mais baixos e similares ao grupo que recebeu o

adjuvante de Freund, sem presença de P10. Estes grupos não apresentaram um

aumento significativo na secreção de TNF-α em relação ao grupo somente infectado

e tratado com dexametasona.

Os grupos de animais que foram tratados com dexametasona e que foram

infectados ou não (Sham), apresentaram uma queda significativa desta citocina,

quando comparados aos animais que não receberam dexametasona.

Resultados

Muñoz J. E

65

Ao observar os animais que não receberam o tratamento com o corticoide, o

grupo de animais infectado apresentou um aumento na secreção de TNF-α

provavelmente, devido ao processo de infecção pelo P. brasiliensis.

Figura 9 - Dosagem da citocina TNF-α pelo método Cytometric Bead Array (CBA)


TNF-

0

500

1000

1500

Sham Infectado Infectado P10 Adj. Itra. Itra+P10 Sulfa+P10Sulfa.Sham

*

***

*

*

*

60 dias 45 dias

pg

/ml


Na figura 10 podemos observar os níveis de secreção de uma das principais

citocinas imunoreguladoras, a IL-10. O homogeneizado dos pulmões apresentou

uma diminuição significativa da IL-10 nos grupos de animais imunizados com P10,

tanto após 45 como 60 dias de infecção. O grupo de animais que recebeu o

adjuvante de Freund, sem peptídeo, mostrou também diminuição na secreção desta

citocina igualmente ao grupo de animais que foi sacrificado 60 dias após a infecção

e que foi tratado somente com itraconazole. Os demais grupos apresentaram níveis

similares ao controle.

α


Resultados

Muñoz J. E

66



IL-10

0

50

100

150

Sham Infectado Infectado P10 Adj. Itra. Itra+P10 Sulfa+P10Sulfa.Sham

*** ***

*

*** **

*

* **

60 dias 45 dias

pg

/ml

* p<0,05 ** p<0,01 *** p<0,0001 quando comparado com os animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste

Tukey.

Com relação à secreção da interleucina 6 (IL-6) nos pulmões dos

camundongos, o grupo de animais anérgicos imunizado com o P10 e também os

animais que receberam o adjuvante de Freund sem P10, apresentaram diminuição

significativa na secreção desta interleucina, após 60 dias de infecção.

Os animais que foram imunizados com o P10, associado ao tratamento

quimioterápico, não apresentaram diferenças significativas na secreção de IL-6, e a

secreção nestes grupos, foi similar à observada nos animais que receberam

somente tratamento com os antifúngicos. Deve-se destacar o aumento na secreção

de IL-6 nos animais (controle) tratados com dexametasona, infectados e sacrificados

com 45 e 60 dias após infecção com Pb18.

Também na figura 11 podemos observar, que na maioria dos grupos,

imunizados ou não com o P10, a secreção desta citocina foi menor aos 45 dias de

infecção. No grupo que não foi infectado e que recebeu o tratamento com o

corticóide (Sham), foi observado uma secreção maior aos 45 dias de sacrificado.

Resultados

Muñoz J. E

67



IL-6

0

200

400

600

Sham Infectado Infectado P10 Adj. Itra. Itra+P10 Sulfa+P10Sulfa.

60 dias45 dias

Sham

****

pg

/ml


** p<0,01 quando comparado com os animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste Tukey.

Outra citocina que foi quantificada é a interleucina 8, e nos protocolos

abordados neste trabalho, não foi possível ver diferença significativa na secreção de

IL-8, nos diferentes grupos de animais imunizados com o peptídeo, como também

nos grupos tratados com as drogas, tanto com 45 como 60 dias após infecção em

relação ao grupo controle infectado e tratado com dexametasona (Figura 12).

O perfil de secreção desta citocina também se mostrou menor na maioria dos

grupos aos 45 dias de infecção, o que demonstra, que no decorrer do tempo de

infecção, o nivel de secreção desta citocina aumenta, podendo cumprir um

importante papel na PCM crônica.

No processo de infecção, esta citocina pode atuar na atração de neutrófilos e

basófilos para o foco da infecção. Porém, se secretada em excesso, pode estar

relacionada com a gravidade da doença.

Resultados

Muñoz J. E

68



IL-8

0

50

100

150

Sham Infectado Infectado P10 Adj. Itra. Itra+P10 Sulfa+P10

60 dias 45 dias

Sulfa.Sham

pg

/ml


Amostras comparadas com os animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste Tukey.

A secreção da quimiocina MCP-1 (monocyte chemoattractant protein 1) pelos

pulmões dos camundongos anérgicos também foi dosada.

A figura 13 indica os valores de MCP-1, onde podemos visualizar uma

diminuição significativa na secreção desta quimiocina, em todos os grupos de

animais, tratados com dexametasona e sacrificados com 60 dias de infecção. Os

camundongos que foram sacrificados com 45 dias de infecção, embora tenham

apresentado diminuição na secreção de MCP-1, esta diminuição não foi significativa

quando comparado ao grupo controle imunossuprimido e infectado.

Os animais imunossuprimidos, mas não infectados (sham), mostraram um

aumento na secreção desta quimiocina, quando comparados aos animais sham que

não foram imunossuprimidos.

Os animais anergicos e infectados apresentaram uma secreção maior desta

quimiocina quando comparados aos animais infectados, que não receberam o

corticóide.

Resultados

Muñoz J. E

69

Figura 13 - Dosagem da quimiocina MCP-1 pelo método Cytometric Bead Array

(CBA) (FACSCalibur, EUA), de homogeneizado dos pulmões de camundongos BALB/c anérgicos, infectados por via intratraqueal com 3x105 células de P. brasiliensis e tratados com as drogas antifúngicas, associadas, ou não, à imunização com P10, e sacrificados com 45 e 60 dias de infecção.

MCP-1

0

200

400

600

800

60 dias 45 dias

Sham Infectado Infectado P10 Adj. Itra. Itra+P10 Sulfa+P10Sulfa.

**

Sham

****** ***

******

pg

/ml


** p<0,01 *** p<0,0001 quando comparado com os animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-testeTukey.

Na figura 14 podemos observar que os animais imunizados com P10 e

sacrificados após 60 dias de infecção, apresentaram diminuição significativa na

secreção de IL-4 Já os animais imunizados com o P10 e sacrificados 45 dias após a

infecção, mostraram dimunição na secreção desta citocina.

Nesta figura pode-se observar que, os grupos de animais imunossuprimidos

apresentaram níveis mais elevados de IL-4, em relação aos grupos de animais não

imunossuprimidos.

Resultados

Muñoz J. E

70



IL-4

0

50

100

150

Sham Infectado Infectado P10 Adj. Itra. Itra.+P10Sulfa. Sulfa.+P10

60 dias 45 dias

Sham

***

**

pg

/ml



A Figura 15 indica os valores de IL-2, onde pode-se observar que os animais

imunizados somente com o P10 apresentaram um aumento significativo na secreção

desta citocina pro-inflamatória. Este aumento foi observado aos 60 e 45 dias após

infecção. Os animais que receberam o tratamento com as drogas, associado à

imunização com o P10, demonstraram um aumento significativo na secreção de IL-

2, após 60 dias de infecção. Com 45 dias de infecção, este aumento não foi

significativo, quando comparado ao grupo imunossuprimido e infectado.

Podemos ressaltar também, que o grupo sham imunossuprimido não

apresentou uma queda representativa na secreção desta citocina, ao comparalo

com o grupo de animais sham não imunossuprimido (Figura 15).

Resultados

Muñoz J. E

71



IL-2

0

100

200

300

Sham Infectado Infectado P10 Adj. Itra. Itra.+P10Sulfa. Sulfa.+P10

60 dias 45 dias

Sham

*

***

**


pg

/ml

* p<0,05 *** p<0,0001 quando comparado com os animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste Tukey.

5.4.1 Padrão Th1/Th2 determinado pela análise do perfil de citocinas

A relação de citocinas Th1/Th2 nos pulmões dos animais imunossuprimidos é

um dado importante, que nos mostra o padrão de resposta imune que está sendo

polarizada após à imunização com o P10. Em conjunto, estes dados sugerem que a

predominância de citocinas do tipo Th1 (IFN- e IL-12) pode ser sufuciente para

controlar o desenvolvimento da doença, conforme verificamos nos grupos de

animais imunizados somente com P10 ou quando este foi associado ao tratamento

quimioterápico (Figura 16).

Esta relação de citocinas (IFN-/IL-10, IFN-/IL-4, IL-12/IL-10, IL-12/IL-4),

também nos mostra que, em vários grupos, a secreção de citocinas pro-inflamatórias

Resultados

Muñoz J. E

72

foi maior nos animais sacrificados com 45 dias de infecção, quando comparados aos

animais sacrificados com 60 dias de infecção.

Figura 16 - Relação do perfil Th1/Th2, pelo método Cytometric Bead Array (CBA) (FACSCalibur, EUA), de homogeneizado dos pulmões de camundongos BALB/c anérgicos.

Estes animais foram infectados por via intratraqueal com 3x105 células de P. brasiliensis

e tratados com as drogas antifúngicas, associadas, ou não, à imunização com P10, e sacrificados com 45 e 60 dias de infecção.

5.5 Detecção por ELISA de anticorpos (IgG Total, IgG2a, IgG2b e IgG1) no soro

de camundongos BALB/c anérgicos infectados intratraquealmente com P.

brasiliensis e imunizados ou não com o Peptídeo P10

5.5.1 Dosagem de IgG total

A alta secreção do anticorpo IgG pode regular a resposta imunológica

mediada por células e pode suprimir ou inibir diretamente a ativação dos linfócitos B

Resultados

Muñoz J. E

73

(por meio da ligação a receptores para FC nos linfócitos B). O impacto da IgG neste

caso é altamente dependente da concentração do anticorpo e da sua afinidade pelo

antígeno, como podemos observar na figura 17, os animais imunizados com o

peptídeo apresentaram uma secreção significativamente maior desta imunoglobulina

quando comparados com os animais controle. Isto está provavelmente relacionado á

regulação da resposta imune no hospedeiro.

Figura 17 – Detecção por ELISA, do isotipo IgG total presente no soro de camundongos BALB/c anérgicos.

IgG Total

Sham N

I

C+ N

I

Sham C+

Adj.

Itra.

Sulfa.

P10

Itra+

P10

Sulfa+P10

0.0

0.5

1.0

1.5

2.0

*****

pg

/ml

de s

oro

Análise, por ELISA, do isotipo IgG total presente no soro de camundongos BALB/c anérgicos, infectados intratraquealmente com Pb18 e imunizados ou não com o P10, após 60 dias de infecção. * significância (p<0,05) relativa aos camundongos somente infectados. Análise por One-way ANOVA seguida de pós-teste Tukey.

5.5.2 Dosagem de IgG2a.

Nós animais imunizados com o peptídeo foi observado um aumento

significativo da imunoglobulina do isotipo IgG2a. Isto devido ao aumento de IFN-,

citocina que regula esse isotipo, assim, demonstrando, uma resposta do tipo Th1

nestes grupos de animais.

Resultados

Muñoz J. E

74

Figura 18 – Detecção por ELISA, do isotipo IgG2a presente no soro de camundongos

BALB/c anérgicos.

IgG2a

Sham

NI

C+

NI

Sham C

+Adj.

Itra.

Sulfa

.P10

Itra+

P10

Sulfa

+P10

0.0

0.2

0.4

0.6

0.8

1.0

**

pg

/ml

de s

oro

Análise, por ELISA, do isotipo IgG2a presente no soro de camundongos BALB/c anérgicos, infectados intratraquealmente com Pb18 e imunizados ou não com o P10, após 60 dias de infecção. * significância (p<0,05) relativa aos camundongos somente infectados. Análise por One-way ANOVA seguida de pós-teste Tukey.

5.5.3 Dosagem de IgG2b e IgG1

A secreção das imunoglobulinas IgG2b e IgG1, (Figuras 19 e 20) nos

camundongos BALB/c anérgicos imunizados com o peptídeo, não apresentou

diferença significativa ao ser comparada com os grupos controle. Isto,

provavelmente, foi devido a resposta imune do tipo Th1, desencadeada nos animais

imunizados com o P10, observada através do aumento na secreção de IFN-γ e IL-

12.

Resultados

Muñoz J. E

75

Figura 19 - Detecção por ELISA, do isotipo IgG2b presente no soro de

camundongos BALB/c anérgicos.

IgG2b

Sham

NI

C+

NI

Sham C

+Adj.

Itra.

Sulfa

.P10

Itra+

P10

Sulfa

+P10

0.0

0.5

1.0

1.5

2.0

2.5

pg

/ml

de s

oro

Análise, por ELISA, do isotipo IgG2b total presente no soro de camundongos BALB/c anérgicos, infectados intratraquealmente com Pb18 e imunizados ou não com o P10, após 60 dias de infecção. Análise por One-way ANOVA seguida de pós-teste Tukey.

Resultados

Muñoz J. E

76

Figura 20 - Detecção por ELISA, do isotipo IgG1 presente no soro de camundongos

BALB/c anérgicos.

IgG1

Sham N

I

C+ N

I

Sham C+

Adj.

Itra.

Sulfa.

P10

Itra+

P10

Sulfa+P10

0.0

0.5

1.0

1.5

2.0

2.5

pg

/ml

de s

oro

Análise, por ELISA, do isotipo IgG1 total presente no soro de camundongos BALB/c anérgicos, infectados intratraquealmente com Pb18 e imunizados ou não com o P10, após 60 dias de infecção. Análise por One-way ANOVA seguida de pós-teste Tukey.

5.6 Dosagens de óxido nítrico do homogeneizado de pulmão

A geração de óxido nítrico foi observada no sobrenadante de pulmão, de

camundongos anérgicos, infectados intratraquealmente, e imunizados ou não com

P10, associado ao tratamento com as drogas. Foram detectadas concentrações

significativas para todos os grupos imunizados com P10, conforme apresentado na

figura 21. Embora o grupo imunizado com P10, associado ao tratamento com

sulfametoxazol/trimetoprim, tenha apresentado niveis similares ao grupo somente

infectado, este aumento foi significativo, isto devido à alta sensibilidade do aparelho

na detecção do óxido nítrico.

Nos animais imunizados com o peptídeo, que obtiveram o menor número de

UFCs, as concentrações de óxido nítrico mostraram-se elevadas em relação aos

demais grupos. Destacam-se os animais imunossuprimidos e infectados que

apresentaram níveis baixos de óxido nítrico, igual ao grupo sham não infectado.

Resultados

Muñoz J. E

77

Pode-se observar nesta figura, que a imunização com P10 promoveu uma

recuperação do sistema gerador de nitrito, o qual é fundamental para que os

macrófagos possam executar sua função na resposta imunológica do hospedeiro.

Figura 21 - Determinação da produção de óxido nítrico no homogenato de pulmão de camundongos BALB/c anérgicos imunizados ou não com o P10 e tratados ou não com as drogas antifúngicas.

Óxido Nítrico

Sham

Infe

ctad

oAdj.

P10

P10

+Itra

P10

+Sulfa

0

50

100

150

200

***

***

***

NO

uM

A dosagem do óxido nítrico no homogenato foi realizada através do analisador de quimioluminescência (NOATM280, Sievers Inc., USA). As barras indicam a média de três leituras. *** p<0,0001 quando comparado com os animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste Tukey.

5.7 Dosagem de óxido nítrico (NO) do sobrenadante de cultura celular

A geração de óxido nítrico foi observada no sobrenadante de cultura celular

de esplenócitos de camundongos anérgicos, infectados intratraquealmente,

imunizados ou não com P10, e tratados ou não com as drogas. Todas as amostras

foram submetidas ao analisador de quimioluminescência (NOATM280, Sievers Inc.,

USA) para mensuração desse mediador citotóxico. Foram detectadas concentrações

significativas para todos os grupos imunizados com P10, conforme apresentado na

figura 22. Este mesmo resultado não observado nos animais controle infectados e

sham os quais apresentaram níveis de óxido nítrico baixos.

Resultados

Muñoz J. E

78

Os grupos de animais tratados somente com itraconazol e sulfametoxazol-

trimetoprim, também apresentaram níveis elevados de óxido nítrico quando

comparados ao grupo somente infectado.

Os animais infectados e não infectados (sham) submetidos ao tratamento

com o corticóide, apresentaram uma queda significativa na produção de óxido nítrico

o que pode demonstrar, neste modelo, um dos efeitos imunossupressores da

dexametasona.

Figura 22 - Determinação da produção de óxido nítrico no sobrenadante de cultura

celular de esplenócitos de camundongos BALB/c anérgicos imunizados ou não com o P10 e tratados ou não com as drogas antifúngicas.

Sham

Infe

ctad

o

Sham

Infe

ctad

o

Freund

Itra.

Sulfa

.P10

Itra+

P10

Sulfa

+P10

0

5

10

15

20

25

*

***

NO

(u

M)

*


A dosagem do óxido nítrico no homogenato foi realizada através do analisador de quimioluminescência (NOATM280, Sievers Inc., USA). As barras indicam a média de três leituras. * p<0,05 *** p<0,0001 quando comparado com os animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste

Tukey.


peroxidase

No tecido pulmonar dos animais infectados com Pb18, podem ser observadas

lesões com granulomas bem definidos, constituídos por agregados de macrófagos,

Resultados

Muñoz J. E

79

células epitelióides, células gigantes multinucleadas, linfócitos e células

polimorfonucleares, ao redor de células fúngicas leveduriformes. Em nosso modelo

experimental, estas populações celulares se encontram diminuídas pelo efeito da

dexametasona.

No ensaio de imunohistoquímica utilizou-se tecido pulmonar dos

camundongos BALB/c anérgicos, para se observar a quantidade e a distribuição de

diversas células, através da ligação de anticorpos a receptores específicos para

identificação destas células.

Como podemos observar na figura 23, os animais imunizados com o P10

(Figura 23B) apresentaram uma marcação mais intensa, especifica ao anticorpo

anti-mouse CD11b, o qual é expresso principalmente por macrófagos. Com o

aumento destas células, também é possível observar um número menor de células

leveduriformes nos animais que receberam o P10. No grupo controle, só infectado e

anérgico, podemos observar mais leveduras e um número menor de células

marcadas (Figura 23A).

Figura 23 – Identificação de células CD11b+ através de imunohistoquímica dos

pulmões dos camundongos BALB/c anérgicos infectados com Pb18 e imunizados ou não com o P10.

(A) Controle positivo (somente infectado) e tratado com PBS, 60 dias após a infecção. (B) Animais infectados e imunizados com P10. Cortes submetidos à marcação pelo anticorpo CD11b e contracoloração com Hematoxilina de Mayer, 10X. As setas pretas indicam os macrófagos, as vermelhas leveduras.

Resultados

Muñoz J. E

80

Na figura 24, podemos observar a marcação de celulas Ly-6G/Ly-6C+,

receptor geralmente mais expresso em neutrófilos, com uma coloração marrom

intensa, onde os animais imunizados com o P10 apresentaram um número maior

destas células (Figura 24 B1 e 24 B2). Também pode ser observado, que as células

Ly-6G/Ly-6C+ ficam aglomerados ao lado das leveduras como indicam as setas

vermelhas. Os animais imunizados com o P10 quando apresentavam leveduras,

estas estavam contidas em granulomas compactos.

O grupo controle, composto de animais imunossuprimidos e infectados

(Figura 24 A1 e 24 A2), mostraram um número menor destas células, assim como

foram observadas muitas células leveduriformes disseminadas pelo tecido e

formação de granulomas frouxos.

O aumento no número de celulas Ly-6G/Ly-6C+ nos animais imunizados com

o P10, foi significativo quando comparado ao grupo de animais não imunizado

(Figura 26).

Figura 24 - Identificação de células Ly-6G/Ly-6C+ através de imunohistoquímica dos pulmões dos camundongos BALB/c anérgicos infectados com Pb18 e imunizados ou não com o P10.

(A1 e A2) Controle, animais somente infectados e imunossuprimidos e sacrificados 60 dias após a infecção. (B1 e B2) Animais infectados, imunossuprimidos e imunizados com P10. Cortes submetidos à marcação pelo anticorpo Ly-6G/Ly-6C e contracoloração com Hematoxilina de Mayer, 10X. As setas vermelhas indicam as células Ly-6G/Ly-6C+ e as amarelas indicam as leveduras.

Resultados

Muñoz J. E

81

Como podemos observar na figura 25 os animais imunossuprimidos e

imunizados com o P10 apresentaram um aumento no numero de células L3T4+,

dentro destas células encontramos a subpopulação de linfócitos T maduros

(incluindo células T “herper”, células T MHC-II restritas e um subtipo de células

NKT), este aumento observado nos pulmões dos animais imunizados com o

peptídeo demonstra que o P10 incrementa a resposta específica contra o fungo,

tornando-se um fator importante no controle da doença.

Ao quantificar o número de células marcadas no tecido pulmonar,

observamos que este aumento de células L3T4+ foi significativo, quando comparado

ao grupo de animais somente infectado (Figura 25).

Figura 25 - Identificação de células L3T4+ através de imunohistoquímica dos

pulmões dos camundongos BALB/c anérgicos infectados com Pb18 e imunizados ou não com o P10.

(A) Controle, animais somente infectados e imunossuprimidos e sacrificados 60 dias após a infecção. (B) Animais infectados, imunossuprimidos e imunizados com P10. Cortes submetidos à marcação pelo anticorpo L3T4 e contracoloração com Hematoxilina de Mayer, 10X. As setas vermelhas indicam as células L3T4+ e as amarelas indicam as leveduras.

Resultados

Muñoz J. E

82

Figura 26 – Quantificação de células do tecido pulmonar de camundongos BALB/c

anérgicos infectados com Pb18 e imunizados ou não com o P10, marcadas pelo método de imunohistoquímica com os anticorpos Ly-6G/Ly-6C e L3T4.

Infe

ctado

P10

Infe

ctado

P10

0

100

200

300

400

500**

*

Ly-6G/Ly-6C

L3T4

No d

e cé

lula

s/m

m2

Grupo infectado, animais somente infectados e imunossuprimidos. Grupo P10 animais infectados, imunossuprimidos e imunizados com P10, os dois grupos foram sacrificados 60 dias após a infecção. * p<0,05 ** p<0,01 quando comparado com os animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA

seguida de pós-teste Tukey.

5.9 Ensaio de proliferação celular

Os linfócitos dos camundongos imunossuprimidos e somente infectados

(Controle) não mostraram proliferação significativa quando comparada com o grupo

não infectado (sham). Entretanto, mesmo após 60 dias da infecção, os linfócitos

esplênicos dos animais desafiados que haviam sido imunizados somente com P10 e

o peptídeo associado as drogas sulfametoxazol/trimetoprim e itraconazol,

apresentaram intensa proliferação, quando estimulados com o peptídeo P10 in vitro,

comparando-se com o grupo controle (sham). Além disso, as células provenientes

de animais anérgicos, que foram imunizados com adjuvante de Freund foram

significativos, proliferando em resposta ao peptídeo P10 de forma significativa

quando comparado com o grupo controle (sham), conforme é observado na figura

27.

Resultados

Muñoz J. E

83

Figura 27 - Ensaio de proliferação celular utilizando linfócitos esplênicos de animais

imunizados com o P10 ou não imunizados 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis e animais controle (sham).

Linfoproliferação

Contr

ole

ConA

Sham

Infe

ctad

oAdj.

P10

P10

+Itra

P10

+Sulfa

0.0

0.1

0.2

0.3

0.4

0.5

*** ******

**

D.O

. 590

As células permaneceram sob estímulo do peptídeo P10 ou Concanavalina A por 144 horas. ** p<0,01 *** p<0,0001 quando comparado ao grupo controle (sham).

5.10 Dosagem de citocinas do sobrenadante de cultura celular

Antes da marcação das células observadas no ensaio de proliferação celular foi

coletado o sobrenadante de cultura das células provenientes do baço de animais

imunizados com o peptídeo ou animais infectados, não imunizados; as quais foram

estimuladas com o peptídeo P10 por 144 horas. No sobrenadante de cultura celular

dos animais imunizados com o peptídeo P10, podemos observar um aumento

significativo de IFN- γ e IL-12, dois importantes citocinas pró-inflamatórias que atuam

na efetiva resposta do hospedeiro contra fungo o que demonstra a atividade do

peptídeo em induzir resposta imune do tipo CD4+ Th1.

Os animais que receberam o tratamento quimioterápico, não apresentaram

aumento de IFN-γ. A secração desta citocina nos esplenócitos de grupos controle foi

similar aos de animais imunossuprimidos e não imunossuprimidos, isto indica que

mesmo tratados com dexametasona, as células de cultura continuam com a

capacidade de produzir citocinas pró-inflamatórias, como IFN-γ.

Resultados

Muñoz J. E

84

Desta forma o tratamento com dexametasona reduz o numero de leucócitos

levando a um estado de imunossupressão, mas não interfere na secreção de

citocinas como pode ser observado na figura 28.

Figura 28 - Dosagem de IFN-γ, pelo método de ELISA em sobrenadante de cultura

de linfócitos esplênicos provenientes de animais imunizados com o P10, 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis.

IFN-

Sham

Infe

ctad

o

Sham

Infe

ctad

oAdj.

Itra.

Sulfa

.P10

Itra+

P10

Sulfa

+P10

0

2000

4000

6000

8000

10000

****


** *

pg

/ml

de s

ob

ren

ad

an

te

* p<0,05 ** p<0,01 quando comparado com os esplenocitos de animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste

Tukey.

Na figura 29 pode-se observar que os níveis de IL-12 secretados pelos

esplenócitos de animais imunizados com o P10 associado ou não ao tratamento

quimioterápico, foram significativamente maiores do que os níveis secretados pelos

esplenócitos de animais não imunizados.

Os animais que foram tratados somente com as drogas e com o adjuvante de

Freund, não apresentaram aumento desta citocina pró-inflamatória (Figura 29).

Resultados

Muñoz J. E

85

Figura 29 - Dosagem de IL-12, pelo método de ELISA em sobrenadante de cultura


IL-12

Sham

Infe

ctad

o

Sham

Infe

ctad

oAdj.

Itra.

Sulfa

.P10

Itra+

P10

Sulfa

+P10

0

50000

100000

150000

**

* *


pg

/ml

de s

ob

ren

ad

an

te

* p<0,05 ** p<0,01 quando comparado com os esplenocitos de animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste

Tukey.

Um resultado que chamou a atenção foi o aumento na secreção da

interleucina IL-10 (IL-10) no sobrenadante de cultura celular, de esplenócitos de

animais imunizados com o P10. Já que esta é uma citocina anti-inflamatória

antagônica ao IFN-γ. Contudo, a IL-10 tem um importante papel regulador no

desenvolvimento da resposta imune “T helper-cell”.

O aumento na secreção desta citocina pode ser devido ao acréscimo na

população de células Treg, que foi observado nos grupos de animais imunizados

com o peptídeo (Figura 37 e 38), lembrando que esta população de células produz

principalmente TGF-β e IL-10 para manter o equilíbrio na resposta imune do

hospedeiro, evitando assim, os efeitos deletereos no tecido devido à uma intensa

resposta imune.

Resultados

Muñoz J. E

86

Figura 30 - Dosagem de IL-10, pelo método de ELISA em sobrenadante de cultura


IL-10

Sham

Infe

ctad

o

Sham

Infe

ctad

oAdj.

Itra.

Sulfa

.P10

Itra+

P10

Sulfa

+P10

0

20000

40000

60000

80000

100000

***

***

***


pg

/ml

de s

ob

ren

ad

an

te

*** p<0,0001 quando comparado com os esplenocitos de animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste Tukey.

Com relação à citocina IL-4, a qual está associada ao padrão Th2 da resposta

imunológica, não foi observada diferença estatística na secreção desta citocina entre

os grupos imunizados com P10 e os grupos não imunizados.

Além disso, pode-se perceber um padrão diferente ao observado no

homogenato de pulmão, onde os animais imunossuprimidos, mesmo não infectados,

mostraram maiores índices desta citocina. No sobrenadante de cultura celular

podemos observar que os animais não imunossuprimidos apresentaram maior

secreção desta citocina, quando comparado aos demais grupos de animais que

receberam dexametasona.

Resultados

Muñoz J. E

87

Figura 31 - Dosagem de IL-4, pelo método de ELISA em sobrenadante de cultura de

linfócitos esplênicos provenientes de animais imunizados ou não com o P10, 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis.

IL-4

Sham

Infe

ctad

o

Sham

Infe

ctad

oAdj.

Itra.

Sulfa

.P10

Itra+

P10

Sulfa

+P10

0

50

100

150


pg

/ml

de s

ob

ren

ad

an

te

Análise por One-way ANOVA seguida de pós-teste Tukey.

Na dosagem de outras importantes citocinas e pouco estudadas na PCM,

encontramos um aumento significativo na secreção da interleucina 18 (IL-18), no

sobrenadante de cultura celular dos camundongos anérgicos, infectados com Pb18

e imunizados com o peptídeo P10 (Figura 32). O aumento na secreção desta

citocina pode ser muito importante no controle da doença, já que a IL-18 é uma

citocina pro-inflamatória da superfamília das IL-1, com um importante papel na

resposta imune inata e adquirida.

Em nosso modelo experimental de anergia, observamos que os animais

imunizados com o P10, apresentaram uma menor carga fúngica nos pulmões e

aumento na secreção de citocinas como IL-12 e IFN-, também apresentaram uma

diminuição na diseminação da doença para outros orgãos como fígado e baço. No

entanto foi observado um aumento significativo na secreção de IL-18, o que

Resultados

Muñoz J. E

88

provavelmente ajuda no desenvolvimento de uma resposta pro-inflamatória em

resposta à infecção pelo fungo.

Também na figura 32 podemos observar que os animais que receberam

dexametasona tiveram uma queda na secreção desta citocina, comparado com os

animais não imunossuprimidos.

Figura 32 - Dosagem de IL-18, pelo método de ELISA em sobrenadante de cultura de linfócitos esplênicos provenientes de animais imunizados com o P10, 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis.

IL-18

Sham

Infe

ctad

o

Sham

Infe

ctad

oAdj.

Itra.

Sulfa

.P10

Itra+

P10

Sulfa

+P10

0

5000

10000

15000


pg

/ml

de s

ob

ren

ad

an

te

* p<0,05 quando comparado com os esplenocitos de animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste Tukey.

Outra importante citocina dosada é a IL-1β a qual tem como principal

atividade, atuar como mediadora da inflamação.

Como podemos observar na figura 33, os animais imunizados somente com

P10 e o grupo que foi imunizado associado ao tratamento com

sulfametoxazol/trimetoprim, tiveram um aumento significativo na secreção de IL-1β,

o que está provavelmente relacionado com o aumento significativo na secreção de

TNF-α, observado nos camundongos imunizados com o P10. Um mecanismo natural

*

Resultados

Muñoz J. E

89

de indução de células produtoras de IL-17 é a presença de IL-6 e IL-1β no local da

infecção, assim o aumento observado na secreção de IL-17 (Figura 34), pode estar

relacionado de forma direta ou indireta com o aumento na secreção de IL-1β, nos

animais imunizados com o P10, o que desencadeia uma via de resposta pró-

inflamatória.

Figura 33 - Dosagem de IL-1β, pelo método de ELISA em sobrenadante de cultura de linfócitos esplênicos provenientes de animais imunizados com o P10, 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis.

IL-1β

Sham

Infe

ctad

o

Sham

Infe

ctad

oAdj.

Itra.

Sulfa.

P10

Itra+P10

Sulfa+P10

0

500

1000

1500

2000

2500

***


pg/m

l de

sobr

enad

ante

* p<0,05 ** p<0,01 quando comparado com os esplenócitos de animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste

Tukey.

A subpopulação de células Th17 é definida pela produção da família de

citocinas IL-17, sendo a IL-17A a mais produzida, seguida por IL-17F, B, D, C, e E;

as células T ativadas são a principal fonte de secreção destas citocinas.

Para a PCM, não esta bem claro o papel da IL-17 no processo de infecção.

No nosso modelo experimental de anergía, observamos um aumento na secreção

desta citocina, para os animais imunizados somente com P10 (Figura 34), sendo

que os grupos que receberam o peptídeo, associado ao tratamento quimioterápico

não apresentaram secreção significativa desta citocina, quando comparado ao grupo

controle imunossuprimido e infectado.

Resultados

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90

O aumento desta citocina pode estar relacionado também com o aumento de

células Ly-6G/Ly-6C e L3T4+ nos pulmões dos camundongos anérgicos, imunizados

com o P10, como podemos observar na marcação específica por imunohistoquimica

nas figuras 24 e 25.

Figura 34 - Dosagem de IL-17A, pelo método de ELISA em sobrenadante de cultura de linfócitos esplênicos provenientes de animais imunizados com o P10, 60 dias após a infecção intratraqueal com 3x105 leveduras de P. brasiliensis.

Sham

Infe

ctados

Sham

Infe

ctados

Adj.Itr

a.

Sulfa.

P10

Itra+P10

Sulfa+P10

0

500

1000

1500

2000


**

pg/m

l de

sobr

enad

ante

** p<0,01 quando comparado com os esplenocitos de animais infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste Tukey.

5.11 Caracterização de células T com fenótipo de memória

A análise da população de células com fenótipo de memória foi realizada

utilizando os esplenócitos após 144h de cultura, estimulados ou não com o P10,

utilizando-se anticorpos anti-CD4/CD44, específicos para marcação de células de

memória em camundongos (Figura 35 A, B, C e D). O gráfico da figura 35 A mostra

que cerca de 3.81% das células presentes, co-expressam as moléculas CD4/CD44.

Por outro lado, foram detectadas quantidades significativas de células com

fenótipo de memória expressando as moléculas CD4/CD44, no grupo de animais

somente imunizados com o P10 (Figuras 35 B). Os resultados obtidos nos grupos de

IL-17A

Resultados

Muñoz J. E

91

camundongos tratados com as drogas antifúngicas itraconazol e sulfametoxazol-

trimetoprim e imunizados com o P10 não mostraram aumento no número de células

com fenótipo de memória. Isto ocorre provavelmente, devido a uma diminuição

significativa da quantidade de leveduras, já que uma menor exposição ao antígeno

induz a um número menor de células T efetoras e de memória. Lembrando que a

resposta por estes dois tipos de células depende da duração e nível de estimulação

antigênica.

Figura 35 - Percentual de linfócitos esplênicos com fenótipo de memória.

Esplenócitos de camundongos controle (sham) A; animais imunizados com o peptídeo (P10) B; animais imunizados com o peptídeo e tratados com Itraconazole C; animais imunizados com o peptídeo e tratados com Sulfametoxazole-trimetoprim D; as amostras foram analisadas após ensaio de proliferação celular. Resultados representam um experimento com pool de três animais por grupo.

B A

C D

Resultados

Muñoz J. E

92

Figura 36 - Percentual de linfócitos esplênicos com fenótipo de memória em


Células TCD4+CD44+

Sham P

10

Itra.

+P10

Sulfa

.+P10

0

2

4

6

*

% c

élu

las T

CD

4+

CD

44+

O percentual de células de memória foi determinado 60 dias após a infecção intratraqueal com 3x10

5 leveduras de P. brasiliensis, as amostras foram analisadas

após ensaio de proliferação celular. Resultados representam um experimento com pool de três animais por grupo. * P < 0,05 quando comparado com animais sham não infectados, não imunizados e tratados com dexametasona. Análise por One-way ANOVA


5.12 Caracterização de células T com fenótipo de reguladoras (Treg)

No modelo experimental de paracoccidioidomicose no estado de

imunossupressão, os resultados também mostram um maior percentual de células T

CD4+Foxp3+ nos animais controle-positivo (apenas infectados com P. brasiliensis,

sem ser imunizados com o peptídeo) quando comparados aos animais controle

(sham).

Resultados

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Figura 37 - Percentual de linfócitos esplênicos com fenótipo de reguladora em


O percentual de linfócitos esplênicos com fenótipo de reguladora foi determinado 60 dias após a infecção intratraqueal com 3x10

5 leveduras de P. brasiliensis, as amostras

foram analisadas após ensaio de proliferação celular. Resultados representam um experimento com pool de três animais por grupo.

A

C

B

D

Resultados

Muñoz J. E

94

Figura 38 - Percentual de linfócitos esplênicos com fenótipo de reguladora em


Células TCD4+Foxp3+

ShamP10

Itra+P10

Sulfa+P10

0

1

2

3

* *

*

% c

élu

las

TC

D4+

Fo

xp3+

As células Treg foram observadas 60 dias após a infecção intratraqueal com 3x10

5

leveduras de P. brasiliensis e foram utilizados como controle animais sham. As amostras foram analisadas após ensaio de proliferação celular. Resultados representam um experimento com pool de três animais por grupo. * P < 0,05 ** P < 0,001 quando comparado com animais sham não infectados não imunizados e tratados com dexametasona. Análise por One-way ANOVA seguida de pós-teste Tukey.

5.13 Contagem global de leucócitos

A contagem global de leucócitos do sangue, dos animais imunossuprimidos e

infectados com Pb18, mostrou um aumento significativo dos glóbulos brancos nos

animais imunizados com o P10, após 60 dias de infecção (Figura 39)

Resultados

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Figura 39 – Contagem global de leucócitos do sangue periférico de camundongos

BALB/c anérgicos.

Contr

oleP10

0

500

1000

1500

Leucograma Global

**

Leu

coci

tos

po

r m

m3

A contagem global de leucócitos foi realizada 60 dias após a infecção intratraqueal com 3x10

5 leveduras de P. brasiliensis e foram utilizados como controle animais

somente infectados e não imunizados. ** P < 0,001 quando comparado com animais infectados e tratados com dexametasona não imunizados. Análise por One-way ANOVA


5.14 Curva de sobrevida de camundongos BALB/c anérgicos

Observamos uma melhor avaliação da capacidade protetora da imunização

com o peptídeo P10 através da curva de sobrevida dos camundongos BALB/c

anérgicos e infectados intratraquealmente com P. brasiliensis, onde a associação do

peptídeo com as drogas Itraconazol e sulfametoxazol/trimetoprim levaram os

animais anérgicos a atingir 100% de sobrevida (Figura 40). O grupo de animais

somente infectados (controle) atingiu 100% de mortalidade no octagésimo primeiro

dia após infecção, os dois grupos de animais que foram tratados com 10 mg/kg de

itraconazol e 15-3 mg/kg de sulfametoxazol/trimetoprim, tiveram uma sobrevida

entorno do 40% a 50%. Entretanto, a imunização com o P10 aumentou em 60% a

sobrevivência dos animais. A imunização com o P10 associado ao tratamento com

as duas drogas levou a 100% de sobrevida, demonstrando desta forma o efeito

aditivo da imunização com o peptídeo associado ao tratamento quimioterápico.

Estes resultados obtidos na curva de sobrevida são muito importantes na

estratégia para aperfeiçoar a formulação vacinal do peptídeo P10, o qual possui

propriedades imunoprotetoras bem determinadas no modelo experimental de PCM.

Resultados

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96

Figura 40 - Curva de sobrevivência de camundongos BALB/c anérgicos infectados,

por via intratraqueal, com 3x105 leveduras de P. brasiliensis isolado 18 e imunossuprimidos, com dexametasona.

0 50 100 150 2000

20

40

60

80

100Controle PBS

Itra.

Sulfa.

P10

P10+Sulfa.

P10+Itra.

Dias de Infecção

Cu

rva d

e S

ob

revid

a

(●) representa o grupo controle (PBS) infectado; (■) tratados com Itraconazol (10 mg/kg); (▲) tratados com Sulfametoxazol-trimetoprim (15-3 mg/kg); (▼) Imunizados com o peptídeo P10; (♦) Imunizados com o P10 e tratados com Sulfametoxazol-trimetoprim (15-3 mg/kg); (□) Imunizados com o P10 e tratados com Itraconazol (10 mg/kg). Experimentos foram realizados em duplicatas, apresentando resultados similares. As curvas são diferentes entre si, Long-ranck (Matel-cox) test p<0,05.

5.14.1 Curva de sobrevida de camundongos Knockout iNOS-/-

Os camundongos C57/BL6 Knockout (iNOS-/-) imunossuprimidos

apresentaram 100% de mortalidade com 72 dias de infecção. Os animais

imunizados com o P10 tiveram uma sobrevida de 80% com 200 dias de infecção

(Figura 41). Após este período, os animais que sobreviveram foram sacrificados e as

unidades formadoras de colônia observadas sendo que, os pulmões destes animais,

com 200 dias de infecção, não apresentaram crescimento de leveduras.

Resultados

Muñoz J. E

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Figura 41 - Curva de sobrevivência de camundongos C57/BL6 Knockout

iNOS-/- infectados por via intratraqueal com 3x105 leveduras de Pb18 e imunossuprimidos, com dexametasona.

0 50 100 150 2000

20

40

60

80

100 Infectados

P10

Dias de Infecção

Sob

revid

a (

%)

Os animais foram imunossuprimidos com dexametasona e infectados com 3x105

leveduras de Pb18. () animais foram imunizados P10 e (•) grupo controle, animais infectados que receberam PBS. As curvas são diferentes entre si, Long-ranck (Matel-cox) test p<0,05.

5.15 Histopatologia do pulmão de camundongos anérgicos infectados e

imunizados, ou não, com o peptídeo 10

Na Figura 42, pode ser observada a comparação histopatológica em

hematoxilina/eosina (HE) entre os diferentes grupos estudados. Nos grupos sham

(imunossuprimido e não infectado) (Figura 42A), pode ser observado o pulmão em

seu estado normal. O grupo de animais infectados, não tratados e imunossuprimidos

(Figura 42B), apresentaram parênquima pulmonar muito comprometido, com grande

infiltrado celular e muitos granulomas frouxos, com células fúngicas viáveis em seu

interior, bem como muitas leveduras dispersas pelo tecido pulmonar. O grupo

infectado e imunizado com P10 (Figura 42C) teve a presença de granulomas

compactos e com poucas células fúngicas. O grupo que recebeu o tratamento com

itraconazol, associado à imunização com P10 (Figura 42D), teve uma melhora

importante no tecido pulmonar, haja visto a baixa recuperação de unidades

formadoras de colônia. Isto também foi observado no grupo tratado com

sulfametoxazol-trimetoprim, associado à imunização com o P10, conforme

Resultados

Muñoz J. E

98

apresentado nos gráficos acima descritos. Em todos os grupos foram observadas

características similares, tanto após 45 como após 60 dias da infecção.

Figura 42 - Histopatologia dos pulmões dos camundongos BALB/c anérgicos

infectados com Pb18 e imunizados, ou não, com P10.

(A) Sham (Controle negativo); (B) Controle positivo (somente infectado e tratado com dexametasona); (C) imunizado com P10; (D) Imunizado com P10 e associado à droga, Estes histológicos são de aniamais sacrificados 60 dias após a infecção. Coloração HE, 20X. As setas indicam granulomas e/ou leveduras.

5.16 Identificação da fibrose pulmonar nos animais imunizados com o peptídeo

P10

Para identificar a formação de fibrose no tecido pulmonar dos camundongos

BALB/c infectados e imunossuprimidos, os cortes histológicos foram submetidos à

coloração de Tricom Masson´s e à coloração de Gomori Silver Reticulina com o

propósito de identificar as fibras de colágeno do tipo I e III respectivamente. O grupo

controle, somente infectado, apresentou estrutura pulmonar comprometida, com

muitos granulomas froxos e muitas leveduras no seu interior. A formação destes

granulomas no grupo infectado levou à formação de fibras de colágeno do tipo I nos

pulmões, como pode ser observado na figura 43A.

Foi observada uma marcada diminuição na formação de fibras de colágeno

nos animais que foram imunizados com o peptídeo P10 os quais não apresentaram

Resultados

Muñoz J. E

99

formação de fibras de colágeno do tipo I como pode ser observado na figura 43B

igualmente ao ser associado com o tratamento medicamentoso (Figura 43 E, F) os

animais que receberam só as drogas tiveram uma diminuição da fribrose pulmonar

mas mesmo assim foram observadas alguns pontos com formação de fibras de

colágeno do tipo I (Figura 43 C, D).

Resultados

Muñoz J. E

100

Figura 43 – Detecção da fibrose pulmonar através da histopatologia dos pulmões de

camundongos BALB/c imunossuprimidos e infectados com Pb18, imunizados, ou não, com P10.

(A) Grupo infectado; (B) grupo infectado e imunizado com o peptídeo P10; (C) grupo infectado e tratado com STM; (D) grupo infectado e tratado com ITRA. (E e F) grupos que receberam STM e ITRA associado ao P10 respetivamente. Coloração Masson´s Tricome, 20X. As setas indicam a formação de fibras de colágeno do tipo I.

Como pode ser observado na figura 44 ao comparar o grupo de animais

somente infectados (A) e o grupo de animais que foram infectados e imunizados

com o P10 (B), se observa que a imunização com o P10 levou a uma diminuição

Resultados

Muñoz J. E

101

significativa na formação de fibras de colágeno do tipo I, que causam a fibrose

pulmonar uma importante secuela da PCM que pode levar a morte.

Figura 44 - Comparação da formação de fibras de colágeno do tipo I.

(A) Grupo só infectado; (B) grupo infectado e imunizado com o peptídeo P10. Coloração Masson´s Tricome, 40X. As setas indicam a formação de fibras de colágeno do tipo I.

Para definir a formação de fibrose pulmonar foi necesario também que os

tecidos fossem submetidos à coloração Gomori Silver Reticulina a qual nos ajuda a

identificar a formação de fibras de colágeno do tipo III.

Como pode ser observado na figura 45 os animais imunizasdos com o

peptídeo P10 apresentaram poucas fibras de colágeno do tipo III quando

comparados aos tecidos pulmonares dos animais somente infectados.

Resultados

Muñoz J. E

102

Figura 45 - Comparação da formação de fibras de colágeno do tipo III.

(A) Grupo somente infectado; (B) grupo infectado e imunizado com o peptídeo P10. Coloração Masson´s Tricome, 40X. As setas indicam a formação de fibras de colágeno do tipo III.

5.17 Biodistribuição do conjugado radiomarcado

A pureza radioquímica obtida foi de 92% por cromatografia de camada

delgada. O produto radiomarcado e a impureza 99mTcO2 ficavam no início da fita

(Rf=0) quando metiletilcetona foi utilizado como solvente. Com acetonitrila 50 %, o

produtose desloca (Rf=1) juntamente com a impureza de 99mTc livre na forma de

pertecnetato de sodio´.

Livre de impurezas, a radiomarcação que foi realizada nos camundongos é

mostrada na figura 46.

Figura 46 – Representação esquemática do desenho de um radiofármaco.

Fonte: Liu et al., 2003

Os resultados do estudo de biodistribuição do radiotraçador em animais

BALB/c sadios estão demonstrados em porcentagem de dose injetada por grama

P10

Resultados

Muñoz J. E

103

(%DI/g). Como podemos observar na figura 47, ao aplicar o P10 radiomarcado,

começou a apresentar uma notável captação no rim e nos pulmões no primeiro

tempo de avaliação (5 min), diminuindo em torno de 50% após os 30 min nos rins.

Foi observado também, que o traçador tem uma depuração mais lenta e

captação maior nos pulmões, no tempo de 1h de avaliação. Em outros órgãos

principalmente no baço e fígado, o radiofármaco teve boa captação no tempo de 4 h.

Figura 47 – Biodistribuição do 99mTc-HYNIC-P10 em camundongos BALB/c sadios

O produto HYNIC-P10-99mTc (0,1 mL) com atividade aproximada de 74 MBq (2 mCi) foi administrado aos animais pela via intravenosa (veia caudal). Os animais (n=6) foram sacrificados por deslocamento cervical aos 5, 30, 60, 120, 240 e 360 minutos após a injeção do radioproduto. Setas pretas indicam o tempo de 4 h, seta vermelha o tempo de 1 h.

5.18 Aquisições de imagens cintilográficas

A última etapa do estudo proposto foi a aquisição de imagem cintilográfica

(Figura 48). A partir dela podemos avaliar o comportamento do radiofármaco (neste

caso o peptídeo P10) em um tempo conhecido. Quanto mais avermelhada a

imagem, maior concentração do peptídeo radiomarcado, este resultado é importante

porque nos mostra a biodistribuição do peptídeo P10 nas primeiras horas de

inoculação no organismo, após 30, 60 e 120 min.

Resultados

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104

Como pode-se observar na figura 48, após 30 min da aplicação do peptídeo

este estava em maior concentração na bexiga e no fígado. Após 120 min o P10

ainda pode ser detectado no fígado e nos linfonodos cervicais o que é determinante,

na resposta imune direcionada contra o P. brasiliensis.

Figura 48 – Imagens cintilográficas em camundongos BALB/c 99mTc-HYNIC ligado

no peptídeo P10.

Animais anestesiados 30, 60 e 120 minutos após a aplicação do P10 ligado ao 99mTc-HYNIC. A aquisição de imagem cintilográfica planar foi efetuada em gama-câmara equipada com um colimador de baixa energia e alta resolução, ângulo de 90º e uma matriz de 256 x 256 X 16, durante 180 segundos. Figado (setas vermelhas), bexiga (setas pretas), lifonodo cervical (setas roxas).

5.19 Determinação da atividade citotóxica in vivo do peptídeo P10

No processo de imunização, os animais recebem 20 µg do peptídeo P10.

Para observar se o P10 apresenta algum efeito citotóxico in vivo, os animais foram

imunizados com uma dose 5 vezes maior (100 µg). A administração de uma maior

concentração de P10 não mostrou alterações nos órgãos dos animais como, fígado,

rins, baço e pulmões. Foram escolhidos estes órgãos, principalmente por dois

motivos, primeiro pelo papel metabólico e de filtração do fígado e rins, órgãos que

metabolizam e secretam sustâncias inoculadas. Através da biomarcação, altas

concentrações do peptídeo P10 foram observadas no fígado, mesmo assim o P10

30 min. 60min. 120min.

Resultados

Muñoz J. E

105

não causou alterações neste órgão (Figura 49 B). Nos rins, também não foram

observadas alterações (Figura 49 C).

Foram escolhidos órgãos onde o P10 tem uma alta atividade no processo de

infecção como os pulmões e os baços. Nenhum destes órgãos apresentou

alterações ao receber uma dose alta do P10 (Figura 49 A e D).

Ao observar que o peptídeo P10 não provocou nenhum efeito colateral em

imunizações com doses de 100 ug, podemos descartar a posibilidade que cause

algum efeito colateral em animais imunizados com 20 ug, dose normalmente

utilizada nas imunizações com este peptídeo.

Resultados

Muñoz J. E

106

Figura 49 - Citotoxicidade in vivo do peptídeo P10.

O peptídeo foi aplicado na concentração de 100µg do peptídeo, pela via i.p. 3 vezes a cada 24 h, na ausência de adjuvante. No quinto dia os animais foram sacrificados e dissecados os órgãos (A) Pulmão, (B) Figado, (C) Rins e (D) Baço, posteriormente foi realizada a coloração de H&E.

5.20 Determinação da atividade citotóxica in vitro do peptídeo P10

Para os ensaios de citotoxicidade in vitro do peptídeo P10, células das

linhagens, endotelial (Human Umbilical Vein Endothelial Cells) HUVEC (5 x 103) e

Resultados

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fibroblasto murino MEFs (mouse embryonic fibroblasts), (1 x 104) foram estimuladas

com 100, 80, 60, 40 e 20 µg/mL de P10 por 24 h. Visivelmente as células

estimuladas com P10 não apresentavam diferencias ao ser comparadas com as

células que não foram estimuladas, para confirmar este resultado foi realizada

contagem através do método de exclusão de Trypam Blue e efetivamente não foi

observada uma diminuição significativa no número de células ao acrescentar o

estimulo com o P10 (Figura 50).

Figura 50 - Citotoxicidade in vitro do peptídeo P10.

0

5

10

15

20

Controle 20ug 40ug 60ug 80ug 100ug

No d

e c

élu

las (

10

3)

Fibroblasto murinoEndotelial humana

Citotoxicidade de diferentes concentrações de P10, avaliada in vitro nas culturas celulares das linhagens endotelial humana (Human Umbilical Vein Endothelial Cells) HUVEC (5x10

3) e fibroblasto murino (mouse embryonic fibroblasts) MEFs (1x10

4), foi

realizado o teste de ANOVA o qual não mostrou diferença significativa entre os grupos.

Discussão

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6 DISCUSSÃO

Diferentes estudos tem demonstrado o papel imunoestimulador do peptídeo

P10 na PCM experimental. O P10 (QTLIAIHTLAIRYAN) da gp43, tem se tornado um

forte candidato vacinal, considerando que este peptídeo leva a uma proliferação de

células TCD4+, polarizando a resposta imune do tipo Th1, caracterizada pelo

aumento na secreção de IFN-γ, que tem a função de estimular à formação de

granulomas que podem conter as leveduras patogênicas (BRUMMER et al., 1988).

O IFN-γ é capaz de ativar os macrófagos pulmonares, os quais tem sua atividade

fungicida aumentada e, portanto, está envolvido na resistência à infecção por P.

brasiliensis. Sua ausência contribui para determinar a suscetibilidade à infecção

(BRUMMER et al., 1988; KASHINO et al., 2000; SOUTO et al., 2000).

A defesa contra as infecções fúngicas exige uma boa interação entre a

resposta imune inata e adaptativa, desde que nenhuma delas seja exacerbada como

foi observado por Pina et al. (2008), onde uma eficiente resposta inata está

associada com suceptibilidade à PCM, no modelo experimental.

Nossos resultados demonstraram através de imunohistoquímica um aumento

de células CD11b+, Ly-6G/Ly-6C+ e L3T4+, nos pulmões dos animais anérgicos

imunizados com o P10. Entre as células que experessam estes receptores

encontramos macrófagos, neutrófilos e Linfócitos TCD4+ respectivamente, os

macrófagos, juntamente com os linfócitos T auxiliares, são os tipos celulares que

mais produzem citocinas. Estes ativam uma rede de células e, entre as numerosas

respostas fisiológicas, está a indução à resposta inflamatória e à proliferação celular

(GOLDSBY et al., 2002).

Entre estas citocinas encontramos o IFN-, fundamental na resposta imune,

contra vários patógenos, já que estimula e/ou ativa os macrófagos, os quais

apresentam atividade fungicida contra conídios e leveduras do P. brasiliensis (CANO

et al., 1998). O TNF-α, outra importante citocina pró-inflamatoria, ajuda na formação

de granulomas para conter o fungo. O aumento desta citocina no processo de

infecção por P. brasiliensis foi observado em estudos experimentais e clínicos

(CALVI et al., 2003).

Ao imunizar os animais com o P10 observamos aumento de IL-12 e IFN-γ tanto

no homogenizado de pulmão como no sobrenadante de cultura celular, estas duas

Discussão

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109

citocinas pro-inflamatórias, atuam na efetiva resposta do hospedeiro contra fungo, o

que demonstra a atividade do peptídeo em induzir resposta imune do tipo CD4+ Th1

produtor de IFN-γ (TABORDA et al., 1998).

A IL-8 é um potente quimiotático e ativador de neutrófilos (MONTON et al.,

1998). Esta citocina é produzida por uma enorme variedade de células, entre elas

podemos destacar os monócitos, linfócitos, células do endotélio ou epitélio e

fibroblastos, em resposta a diferentes estímulos como lipopolissacarídeos e a outras

citocinas (TNF-α, IL1-β). Embora nossos resultados não mostrem diferença

significativa na secreção de IL-8 por ELISA, comseguimos observar por meio de

imunohistoquimica, um recrutamento e aumento de neutrófilos no foco da infecção.

A quimiocina MCP-1 (monocyte chemoattractant protein 1) atua ligando-se à

proteína G, presente nos leucócitos, para sua posterior ativação e migração destas

células. Chensue et al. (1996), examinaram culturas de macrófagos, isolados de

granulomas e nódulos linfáticos, revelando um aumento na produção da MCP-1

principalmente, quando o tipo de resposta imune é Th2. Este mesmo grupo de

pesquisadores demonstrou que esta citocina ainda inibe a produção de IL-12, por

macrófagos in vivo e in vitro, sendo que IL-12 é uma das citocinas pró-inflamatórias

de grande importância, juntamente com IFN-γ na resposta celular – Th1.

O aumento na secreção destas citocinas estimula toda uma cascata na

resposta imune, ativando outros leucócitos que secretam outras citocinas e assim

sucesivamente. Nos animais imunizados com o P10 encontramos aumento

significativo de citocinas pro-inflamatórias como a IL-1β que é produzida por

monócitos e macrófagos e sua ação mais importante na inflamação, se deve aos

efeitos no endotélio, ativação de leucócitos e fibroblastos, bem como a indução das

reações da fase aguda. Esta citocina atua na regulação da leucopoiese durante um

processo infeccioso. A IL-1 β pode estimular a liberação de fatores de crescimento

(CSF) para granulócitos, macrófagos e monócitos. Além disso, estimula a liberação

do fator de necrose tumoral (TNF) que estimula os linfócitos T (OKAMURA et al.,

1998).

A IL-18 é uma interleucina pouco estudada na PCM e que mostrou um

aumento significativo nos animais imunizados com o P10, é secretada por

macrófagos ativados, linfócitos T e B e células dendríticas (OKAMURA et al., 1995;

OKAMURA et al., 1998). A principal função da IL-18 é induzir a proliferação dos

Discussão

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linfócitos Th1 e potencializar a produção de IFN-, aumentando assim a secreção de

IL-12 (ROBINSON et al., 1997; YOSHIMOTO et al., 1998). A super expressão desta

citocina pode promover uma rápida cura da infecção, mas pode levar a uma

resposta imune exagerada, com danos teciduais na Paracoccidioidomicose

(CORVINO et al., 2006).

Os camundongos imunossuprimidos e imunizados somente com o P10,

apresentaram queda na secreção de IL-6 e os gupos de animais imunizados com o

peptídeo associado ao tratamento quimioterápico não mostraram alterações na

secreção desta citocina. Quando estudamos as funções da IL-6, encontramos que

ela pode contribuir para o crescimento e/ou proliferação de linfócitos B, mas também

pode ajudar no estímulo da unidade formadora de colônias de granulócitos-

macrófagos (CSF-GM), o qual pode chegar a reverter o estado de imunossupressão

por dexametasona (ROILIDES et al, 1996).

Nossos resultados mostraram uma diminuição significativa na secreção de

algumas citocinas anti-inflamatórias nos animais imunizados com o P10, no

homogeneizado de pulmão, o que no demonstra a prevalência do perfil Th1.

No sobrenadante de cultura celular também foi observado um aumento

significativo de algumas citocinas pro-inflamatórias como IFN-γ e IL-12. No entanto,

houve aumento na secreção da IL-10, citocina imunoreguladora e antagonista do

IFN-γ, este resultado não esperado após 144 horas de cultura dos esplenócitos.

Contudo, a IL-10 tem um importante papel regulador no desenvolvimento da

resposta imune “T helper-cell” e resposta imune inata (HUFFNAGLE; DEEPE 2003;

MENCACCI et al., 2000; ROMANI, 2004).

O aumento na secreção de IL-10 pode ser devido ao pequeno mas,

significativo aumento na população de células Treg, que foi observado nos grupos

de animais imunizados com o peptídeo, lembrando que esta população de células

produz principalmente TGF-β e IL-10 para manter o equilíbrio na resposta imune do

hospedeiro, evitando assim, o dano no tecido (BELKAID, 2008).

Animais imunizados somente com P10 apresentaram um aumento na

secreção de IL-17A. Tem sido descrito, que as células T CD8+ de memória,

neutrófilos, monócitos e linfócitos T CD4+, são células produtoras destas citocinas

principalmente IL-17A (FERRETTI et al., 2003; ZHOU et al., 2005). A IL-17 é uma

citocina pro-inflamatória, importante no “clearance” de patógenos extracelulares e a

Discussão

Muñoz J. E

111

deficiência desta citocina pode aumentar a susceptibilidade à infecção, como foi

descrito em Candida albicans (HUANG W et al., 2004).

Os resultados obtidos nos grupos de camundongos tratados com as drogas

antifúngicas itraconazol e sulfametoxazol-trimetoprim e imunizados com o P10 não

mostraram aumento no número de células com fenótipo de memória. Isto pode

acontecer devido a uma diminuição significativa do patógeno, já que uma menor

exposição ao antígeno induz um número menor de células T efetoras e de memória.

Lembrando que a resposta por estes dois tipos de células depende da duração e

nível de estimulação antigênica.

Nos animais que foram imunossuprimidos e sacrificados com 60 dias de

infecção e que foram imunizados com o P10, foi possível observar um aumento

significativo de células Ly-6G/Ly-6C+ (Figura 26), este receptor é principalmente

expresso pelos neutrófilos. As primeiras características histopatológicas de

pacientes com PCM mostram uma forte evidência do envolvimento dos neutrófilos

na resposta primária do hospedeiro contra o fungo (COSTA et al., 2007). A presença

destas células tem sido detectada no tecido infectado, sangue e medula de

pacientes com paracoccidioidomicose (MARTINEZ, 1993).

As células polimorfonucleares (PMNs), entre as quais encontramos os

neutrófilos, foram observadas nos pulmões de camundongos BALB/c infectados com

Paracoccidioides brasiliensis, até um período de 24 semanas, sendo este número

decrescente ao aumentar o tempo de infecção (McEWEN et al., 1987).

O aumento na secreção de citocinas pro-inflamatórias e na produção de óxido

nítrico nos pulmões dos camundongos BALB/c anérgicos, infectados com o isolado

virulento Pb18, e imunizados com o P10, está direta ou indiretamente relacionado ao

aumento na população das células CD11b+, Ly-6G/Ly-6C+ e L3T4+, nos animais

imunizados.

Os animais imunizados com o P10 tiveram aumento significativo na produção

de óxido nítrico, este gás solúvel é fundamental na resposta imune contra diversos

patógenos e é sintetizado pela óxido nítrico sintase (iNOS). Quando esta enzima é

depletada nos camundongos Knockout para iNOS-/-, conseguimos observar melhor o

papel do óxido nítrico no processo de imunização. Observamos que mesmo os

animais onde a iNOS foi depletada, a imunização com P10 mostrou uma sobrevida

maior do que nos animais não imunizados. Isto significa que o óxido nítrico é

Discussão

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112

importante na resposta ao P. brasiliensis, mas não é fundamental, já que por outras

vias ativadas o sistema imune consegue evitar a infecção causada pelo fungo. Uma

destas posibilidades pode ser a morte do fugo diretamente pelos linfócitos T CD4+,

nos quais foi comprobada a capacidade citotóxica destes, atuando diretamente no

patógeno (LEVITZ et al., 1995).

É importante ter um aumento na resposta inflamatória, para conter a infecção

e disseminação do fungo no hospedeiro, mas é importante também evitar os efeitos

deletérios por uma resposta imune exacerbada. A fibrose pulmonar é uma severa e

progressiva seqüela da PCM. O desenvolvimento da fibrose pulmonar é atribuída a

um prolongado estímulo inflamatório (NARANJO et al., 2010) o qual é comum na

PCM por ser uma doença crônica. Os animais imunizados somente com o P10 ou

associado às drogas, apresentaram uma diminuição da fibrose pulmonar, uma

provável explicação é a conseqüente diminuição da carga fúngica nos pulmões dos

animais imunizados, o que levou a uma queda no estímulo e conseqüente

diminuição da fibrose pulmonar.

Neste trabalho observamos o efeito aditivo da imunização com o P10 no

modelo de anergia na PCM experimental. Verificamos que os animais anérgicos

infectados com o isolado Pb18 e imunizados somente com P10 apresentavam

redução significativa da carga fúngica nos pulmões e menor disseminação da

doença, quando comparados com os animais-controle. Os animais que foram

tratados com as drogas sulfametoxazol/trimetoprim e itraconazol, associadas ao

P10, mostraram 100% de sobrevida por um período de 200 dias e as melhoras

foram evidentes. As características fenotípicas destes animais eram bem diferentes

quando comparados aos animais não imunizados. Avaliando as unidades

formadoras de colônias, constatamos que o P10 foi capaz de diminuir o avanço da

doença para outros órgãos. Além disso, o aumento nos níveis de citocinas, pro-

inflamatórias, nos pulmões, indicando uma restauração da imunidade celular desses

animais. TNF-α, juntamente com IL-12 e IL-18, potencializam a atividade fungicida

de macrófagos. Na PCM, o TNF-α está diretamente relacionado com a formação de

granulomas compactos (MAMONI; BLOTTA, 2006).

A análise histopatológica, com coloração por hematoxilina/eosina (HE), dos

animais anérgicos, mostrou um parênquima pulmonar comprometido, com bastante

infiltrado celular e a coloração de Gomori-Grocott demonstrou um número elevado

Discussão

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113

de leveduras dispersas pelo tecido. Nos animais anérgicos imunizados com o P10

foi observado um parênquima pulmonar mais conservado, com formação de

granulomas pequenos, compactos e com poucas leveduras.

Atualmente, o tratamento da PCM está relacionado à gravidade da doença,

ao tipo de droga utilizada e o tempo de uso. Toxicidade das drogas e relatos de

recidivas, devido à resistência do fungo, tem sido observados, o que torna

importante a pesquisa de novos adjuvantes terapêuticos com o propósito de diminuir

o tempo de tratamento e reduzir as recidivas evitando a disseminação do fungo para

outros sítios anatômicos.

O uso de vacinas preventivas ou terapêuticas em doenças infecciosas tem se

expandido e exige a seleção de componentes imunogênicos que possam levar a

proteção do hospedeiro. Resultados obtidos em nosso laboratório reforçam a

conclusão de que o P10 protege contra a infecção experimental e abre perspectivas

para o estudo em humanos, tornando-se um importante candidato vacinal para ser

testado na paracoccidioidomicose humana. A eficácia da imunização do P10,

associado a outros adjuvantes, ou mesmo sozinho, abre novas perspectivas.

Diferentes formulações vêm sendo testadas com o propósito de encontrar a

melhor proteção na PCM experimental. O peptídeo P10 é um importante candidato

vacinal, o que nos leva a pesquisar cada vez mais a interação deste peptídeo com

outras células e novos adjuvantes que potencializem o efeito protetor deste

peptídeo, como foi observado com a Flagelina (APÊNDICE A, Artigo 4), o uso de

nanoparticulas (APÊNDICE A, Artigo 5), o lipídeo catiônico (APÊNDICE A, Artigo 9)

entre outros.

E quando utilizadas como adjuvante, elas são mais eficientes na

apresentação de um peptídeo, por este motivo foram realizados experimentos com

estas células pulsadas com o peptídeo P10 (MAGALHAES et al., 2011), e foi

observado que, os animais vacinados, apresentaram uma diminuição significativa da

carga fúngica nos pulmões, o que evidencia o potencial das células dendríticas em

atuar como adjuvante na formulação de uma vacina utilizando o peptídeo P10 para o

tratamento e cura da PCM.

Nossos resultados sugerem que o peptídeo P10 é um forte candidato vacinal

que pode atuar como adjuvante ao tratamento medicamentoso, representando uma

Discussão

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114

alternativa promissória na geração de uma vacina anti-PCM e evitando possíveis

recidivas no hospedeiro imunossuprimido.

Conclusões

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115

7 CONCLUSÕES

- Verificamos através da contagem de leucócitos e do teste de hipersensibilidade do

tipo tardia (HTT) que os camundongos BALB/c apresentam estado de anergia a

partir de 30 dias de tratamento com o fosfato de dexametasona.

- Observamos que a imunização com o peptídeo P10, associado ao tratamento

medicamentoso, apresentou um efeito aditivo, levando à redução da carga fúngica e

impediu a disseminação do fungo para outros órgãos como o baço e o fígado dos

animais imunossuprimidos infectados com o isolado virulento Pb18.

- A pesquisa de nitrito, no homogeneizado pulmonar, de camundongos BALB/c

anérgicos, indicou a presença de altos títulos, nos animais imunizados com o P10

quando comparados ao grupo controle.

- Os camundongos anérgicos imunizados com P10 apresentaram um aumento na

secreção de citocinas pro-inflamatórias, como IFN-γ, TNF- e IL-12.

- O P10 foi capaz de manter uma linfoproliferação de TCD4+ elevada em

camundongos infectados com Pb18, em estado anérgico induzido pela

administração de fosfato de dexametasona.

- Análise por imunohistoquímica demonstrou um aumento das populações celulares

CD11b+, Ly-6G/Ly-6C+ e L3T4+ nos pulmões dos animais anérgicos imunizados

com o P10. Entre células que expressam estes marcadores encontramos

macrófagos, neutrófilos e linfócitos TCD4+ respectivamente.

- Verificamos, através dos cortes histológicos, que os pulmões dos animais

imunizados com o peptídeo P10 associado às drogas apresentaram um parênquima

pulmonar conservado, com uma diminuição significante de células fúngicas.

- O efeito aditivo da imunização com o P10 levou a uma sobrevida de 100% dos

animais anérgicos durante um período de 200 dias. Todos os animais (100%) não

imunizados morreram com 80 dias de infecção.

- A biodistribuição do P10 mostrou captação nos pulmões e nos linfonodos cervicais,

nas primeiras horas após imunização.

Conclusões

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116

- Ensaios de citotoxicidade in vitro e in vivo mostraram que o peptídeo P10 não possui

toxicidade em células endoteliais humanas e em fibroblastos murinos, o que torna

este peptídeo uma vacina promissora na PCM.

Referências

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117

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APÊNDICES

APÊNDICE A - Artigos Publicados em Periódicos Indexados Artigo 1 - MUÑOZ, J. E.; LUFT, V. D.; AMORIM, A.; MAGALHÃES, A.; THOMAZ,

L.; NOSANCHUK, J. D.; TRAVASSOS, L. R.; TABORDA C. P. Immunization with P10 peptide increases specific immunity and protects immunosuppressed BALB/c mice infected with virulent yeasts of Paracoccidioides brasiliensis. Microbes and Infection, p. xx, 2013.

Artigo 2 - MARQUES, A.; DA SILVA, M.; JULIANO, M.; MUÑOZ, J. E.;

TRAVASSOS, L. R.; TABORDA, C. P. Additive effect of P10 immunization and chemotherapy in anergic mice challenged intratracheally with virulent yeasts of Paracoccidioides brasiliensis. Microbes and Infection, p. 1, 2008.

Artigo 3 - BUISSA-FILHO, R.; PUCCIA, R.; MARQUES, A. F.; PINTO, F. A.;

MUÑOZ, J. E.; NOSANCHUK, J. D.; TRAVASSOS, L. R.; TABORDA, C. P. The Monoclonal Antibody against the Major Diagnostic Antigen of Paracoccidioides brasiliensis Mediates Immune Protection in Infected BALB/c Mice Challenged Intratracheally with the Fungus. Infection and Immunity, v. 76, p. 3321-3328, 2008.

Artigo 4 - BRAGA, C. J. M.; RITTNER, G. M. G.; MUÑOZ, J. E.; TEIXEIRA, A. F.;

MASSIS, L. M.; SBROGIO-ALMEIDA, M. E.; TABORDA, C. P.; TRAVASSOS, L. R.; FERREIRA, L. C. S. Paracoccidioides brasiliensis Vaccine Formulations Based on the gp43-Derived P10 Sequence and the Salmonella enterica FliC Flagellin. Infection and Immunity, v. 77, p. 1700-1707, 2009.

Artigo 5 - AMARAL, A. C.; MARQUES, A. F.; MUÑOZ, J. E; BOCCA, A. L.;

SIMIONI, A. R.; TEDESCO, A. C.; MORAIS, P. C.; TRAVASSOS, L. R.; TABORDA, C. P.; FELIPE, M.S.S. Poly (lactic acid-glycolic acid) nanoparticles markedly improve immunological protection provided by peptide P10 against murine paracoccidioidomycosis. British Journal of Pharmacology, p. 1-7, 2010.

Artigo 6 - DEGOBBI, C.; LOPES, F. D. T. Q. S.; CARVALHO-OLIVEIRA, R.;

MUÑOZ, J. E.; SALDIVA, P. H. N. Correlation of fungi and endotoxin with PM2.5 and meteorological parameters in atmosphere of Sao Paulo, Brazil. Atmospheric Environment, v. 45, p. 2277-2283, 2011.

Artigo 7 - ROSSI, D. C. P.; MUÑOZ, J. E.; BELMONTE, R.; CARVALHO, D. D.;

FAINTUCH, B.; BORELLI, P.; MIRANDA, A.; TABORDA, C. P.; DAFFRE, S. Therapeutic use of a cationic antimicrobial peptide from the spider Acanthoscurria gomesiana in the control of experimental candidiasis. BMC Microbiology, v. 12, p. 1-12, 2012.

Apêndices

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Artigo 8 - RITTNER, G. M. G.; MUÑOZ, J. E.; MARQUES, A. F.; NOSANCHUK,

J. D.; TABORDA, C. P.; TRAVASSOS, L. R. Therapeutic DNA Vaccine Encoding Peptide P10 against Experimental Paracoccidioidomycosis. Plos Neglected Tropical Diseases, v. 6, p. e1519-9, 2012.

Artigo 9 - MAYORGA, O.; MUÑOZ, J. E.; LINCOPAN, N.; TEIXEIRA, A,;

FERREIRA, L. C. S.; TRAVASSOS, L.; TABORDA C. P. The role of adjuvants in therapeutic protection against paracoccidioidomycosis after immunization with the P10 peptide. Frontiers in Microbiology, 3:154. doi:10.3389/fmicb.2012.00154. 2012.

APÊNDICE B – Artigos no Prelo

1. SANTOS, D. A.; SILVA, F. D.; MUÑOZ, J. E.; SOARES, M. A.; CISALPINO, P. S.; TABORDA, C. P. Paracoccidioides lutzii: exoantigenic profile and immunotherapy based on anti-gp43 monoclonal antibodies and P10 peptide.

2. FONSECA, F. L.; GUIMARÃES, A. J.; DUTRA, F. F.; SILVA, F. D.; MUÑOZ, J. E.; TABORDA, C. P.; BOZZA, M. T.; NIMRICHTER, L.; CASADEVALL, A.; RODRIGUES, M. L. Binding of the wheat germ lectin to Cryptococcus neoformans chitooligomers impairs extracellular polysaccharide release, TLR2-mediated interaction with phagocytes, and brain colonization in mice.

3. MUÑOZ, J. E.; ROSSI, D. C.; ARRUDA, C. A.; NADER, O. M.; CORTE, M. D.; TABORDA, C. P. Inhibitory activity of limonene against isolated virulent for Candida albicans in vitro and in vivo.

APÊNDICE C – Patente.

1. AMARAL, A.; MARQUES, A. F.; MUÑOZ, J. E.; BOCCA, A. L.; SIMIONI, A.

R.; TITZE-DE-ALMEIDA, R.; TEDESCO, A. C.; MORAIS, P. C.; TABORDA, C. P.; TRAVASSOS, L. R.; FELIPE, M. S. S. Formulação nanoestruturada do peptídeo imunoprotetor P10 com ácido dimercaptosuccínico em polímero de ácido polilático-poliglicólico para o tratamento de micoses. 2008.

Artigo 1

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Artigo 1

MUÑOZ, J. E.; LUFT, V. D.; AMORIM, A.; MAGALHÃES, A.; THOMAZ, L.; NOSANCHUK, J. D.; TRAVASSOS, L. R.; TABORDA C. P. Immunization with P10 peptide increases specific immunity and protects immunosuppressed BALB/c mice infected with virulent yeasts of Paracoccidioides brasiliensis. Microbes and Infection, p. xx, 2013.

Immunization with P10 peptide increases specific immunity and protects

immunosuppressed BALB/c mice infected with virulent yeasts of Paracoccidioides

brasiliensis

Julián E. Muñoz 1, Vinicius D. Luft

1, Juliana Amorim

1, Adriana Magalhães

1, Luciana

Thomaz 1, Joshua D. Nosanchuk

2, Luiz R. Travassos

3, Carlos P. Taborda

1,4,#

1 Department of Microbiology, Institute of Biomedical Sciences and

4 Laboratory of Medical

Mycology-LIM53/IMTSP, University of São Paulo, São Paulo, SP, Brazil.

2 Departments of Medicine, and Microbiology and Immunology, Albert Einstein College of

Medicine, Bronx, New York, United States of America.

3 Department of Microbiology, Immunology and Parasitology, Federal University of São

Paulo, São Paulo, SP, Brazil.

# Corresponding author: Department of Microbiology, Biomedical Sciences Institute and

Laboratory of Medical Mycology-LIM53/IMTSP, University of São Paulo, Av. Prof. Lineu

Prestes, 1374, São Paulo, SP 05008-900, Brazil. Tel. +55-11-30917345, e-mail:

[email protected]

Running title: P10 protects anergic mice infected with P. brasiliensis

SUMMARY

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the

dimorphic fungus Paracoccidioides brasiliensis. The acute/subacute forms of the disease are

most severe and are frequently lethal without antifungal treatment. A peptide from the major

diagnostic antigen gp43, named P10, induces a T-CD4+ helper-1 immune response in mice of

different haplotypes and protects against intratracheal challenge with virulent P. brasiliensis.

In the present study, we evaluated the efficacy of the P10 peptide alone or combined with

antifungal drug treatments in mice immunosuppressed with dexamethasone and infected with

a highly virulent human isolate of P. brasiliensis, Pb18. P10 immunization led to an effective

cellular immune response augmenting T cell proliferative responses, and the stimulated

splenocytes produced higher levels of IFN-γ, IL-1β and IL-12 as well as increased NO

concentrations. P10 immunization also led to a minimization in fibrosis in response to Pb18

infection. The addition of antifungal drugs to P10 immunization most significantly impacted

survival after lethal challenge with Pb18 as P10 immunization with the administration of

either itraconazole or sulfamethoxazole/trimethoprim resulted in 100% survival at 200 days

post-infection, whereas untreated mice died within 80 days and P10 alone produced a survival

rate of 60%. The present study shows that P10 immunization promotes a strong specific

immune response even in immunocompromised hosts. P10 is a promising vaccine candidate

for use in immunocompromised hosts.

Key words: P. brasiliensis; Anergy; Chemotherapy; Immunization.

INTRODUCTION

The thermally dimorphic fungus Paracoccidioides brasiliensis is the etiological agent

of paracoccidioidomycosis (PCM). PCM is the most frequent systemic mycosis in Latin

America, with the highest incidence of diagnosis in Brazil, Argentina, Colombia, and

Venezuela (22). The main route of acquisition occurs by the inhalation of fungal particles,

which usually leads to an asymptomatic infection (4). There are two main clinical forms of

PCM, acute/subacute and chronic. The acute and subacute form is characterized by a rapid

course (weeks to months), impaired cellular immunity, an absence of delayed-type

hypersensitivity reactions and a high mortality rate. The chronic form affects mainly adult

males with ages between 30–50 years-old and primarily manifests with predominant

pulmonary and/or mucocutaneous involvement (10).

An effective cellular immune response is essential for the control of experimental and

human PCM (6). Several studies have shown that high levels of specific antibodies and

polyclonal activation of B cells are associated with the severe form of the disease whereas

inflammatory cytokines, such as IFN-γ, IL-12 and TNF-α, have an important protective role

in the host resistance (29, 8). Enhancement of these cytokines elicits a strong cell-mediated

immune response that can combat PCM. Notably, IFN-γ has been used to activate

macrophages, which augments their fungicidal activity against P. brasiliensis (5).

The glycoprotein of 43 kDa (gp43) of P. brasiliensis is the main diagnostic antigen for

PCM as it is recognized by nearly 100% of sera from patients (20). The gp43 binds laminin, a

protein component of the extracellular matrix of mammalian tissues, facilitating fungal

invasion and the subsequent destruction of tissues (32). The gp43 carries an immunodominant

epitope that induces a predominant IFN-γ-mediated Th-1 response (31). It is primarily

responsible for delayed type sensitive (DTH) reactions in infected animals (24). A 15-amino-

acid peptide of gp43 (QTLIAIHTLAIRYAN), designated as P10, presents a CD4 T-cell

specific epitope that is recognized by the molecules of the major histocompatibility complex

class II. P10 is the main epitope in gp43 responsible for activating the cellular immune

response in BALB/c mice (31).

Treatment of PCM requires intensive and prolonged antifungal chemotherapy.

Treatment with sulfonamides (either sulfamethoxazole or sulfadiazine) combined with

trimethoprim, amphotericin B, or azoles is typically administered from 2 to 6 months,

although extended periods of treatment (years) are often necessary depending on the drug

employed and the disease severity (26). Unfortunately, relapses are particularly common in

patients receiving short durations of treatment (13).

Host immune status can increase the risk for and severity of PCM. For example,

severe cases of PCM involving immunosuppressed patients with cancer or after renal

transplantation have been described (27), presumably due to reactivation of a latent lesion in

the setting of cytotoxic drugs. PCM occurs in patients with HIV infection, although PCM has

a significantly lower incidence in patients with HIV infection compared to disease due to

Cryptococcus or Histoplasma (23). Notably, PCM patients with HIV infection frequently

present with rapidly progressive, often multi-focal disease and they also are more likely to

relapse after initial treatment (17) and HIV patients coinfected with P. brasiliensis have less

immunoreactivity to antigens from this fungus (2).

Corticosteroids such as Dexamethasone (DEX) can be used to treat many different

diseases including cancer. DEX is effective reducing airway inflammation through multiples

mechanisms, including the modulation in the synthesis of many inflammatory cytokines (9).

Influence multiple transduction pathways led at immune suppression, condition which

exposes people to contract a fungal disease. Although P. brasiliensis is not an opportunistic

fungus has been observed some cases of infection in patients undergoing treatment with

immunosuppressive drugs. Consequently, the aim of this work is to improve the

chemotherapy treatment through the additive effect induced by the immunization of P10

peptide associated with antifungal drugs in an anergic model of paracoccidioidomycosis (16).

In of the future, to turn the P10 a therapeutic vaccine in human PCM associated with drug

treatment could reduce time to treatment and avoid patient relapses and drug side effects.

MATERIAL AND METHODS

Animals. 5 Male BALB/c mice per group (6 to 8 weeks old), were housed in polypropylene

cages under Specific Pathogen Free conditions and all materials were sterilized prior to use.

Animals used in this study were bred at Institute of Biomedical Sciences of University of São

Paulo animal facility. All experiments involving animals were conducted and approved by

Ethics Committee of Institute of Biomedical Sciences of University of São Paulo and

conducted in accordance with international recommendations.

Immunosuppression of mice. Dexamethasone phosphate (Sigma, St Louis, MO) was used to

induce immune suppression, this corticoid was added to the drinking water of animals twenty

days before infection and treatment remained until the day of sacrifice sixty days post

infection. Assuming an average water intake of 5 ml per day for 30 days, the daily

dexamethasone phosphate dose was calculated as 0.15 mg kg-1 (16). Control animals (non-

Dex) did not receive dexamethasone phosphate.

Fungal strain. Virulent P. brasiliensis Pb18 yeast cells were used to infect the animals. The

fungal strain was maintained by weekly passage on solid Sabouraud medium at 37°C. After 7

to 10 days of growth, yeast cells were cultivated in modified McVeigh-Morton medium at

37°C for 5 to 7 days (21). The fungal cells were then collected, washed in phosphate-buffered

saline (PBS pH 7.2) and counted in a hemocytometer. The viability of fungal suspensions was

determined by staining with trypan blue (Sigma, St. Louis, MO) and was always higher than

90%. The virulence of the Pb18 strain was checked in each experiment by infecting BALB/c

mice i.t. and recovering the yeast cells from the infected organs.

Intratracheal infection. BALB/c mice were inoculated i.t. with 3x105 yeast cells/animal of

virulent P. brasiliensis Pb18 in sterile saline (0.85% NaCl). A maximum volume of 50 μl was

inoculated per mouse. Briefly, the mice were anesthetized i.p. with 200 µl of a solution

containing 80 mg/kg ketamine and 10 mg/kg of xylazine (both from União Química

Farmacêutica, Brazil). After approximately 5 min, their necks were hyperextended, and the

tracheas were exposed at the level of the thyroid and injected with 3x105

yeast cells.

Peptide synthesis and purification. P10 peptide used in this study was purchased from

Peptide 2.0 (Chantilly, VA). HPLC and MS analyses showed that the synthetic P10 was 98%

pure.

Immunization of mice. Anergic BALB/c mice (6- to 8-week old males) were immunized for

with 20 ug of P10 once weekly for four weeks 30 days after i.t. infection. The first

immunization was subcutaneous with P10 in complete Freund’s adjuvant (CFA) and

subsequently peptide immunization was i.p. with incomplete Freund’s adjuvant (IFA).

Control mice were injected with CFA and IFA alone without the presence of the peptide.

Treatment with Antifungal Drugs. Antifungal drug treatment started 30 days after i.t.

infection. Thereafter, mice received doses of 10 mg/kg itraconazole (ITC) (Janssen

Pharmaceutica, NV), or 15-3 mg/kg sulfamethoxazole/trimethoprim (SMT/TMP); (Bac-

sulfitrin, Ducto - Brazil) every 24h for 30 days (to day 60 post infection). All drug

administrations were i.p.

Groups studied. Ten groups of mice (with 6 animals each) were used. Four controls were

included: sham non-DEX, mice that not receive any intervention; infected non-DEX, mice

infected with Pb 18, but not treated with steroids; Sham, uninfected mice immunosuppressed

with dexamethasone phosphate; Infected, immunosuppressed mice infected with Pb18 strain;

CFA/IFA, mice immunosuppressed, infected and immunized with CFA/IFA; P10,

immunosuppressed, infected and immunized with P10 peptide; (ITC), immunosuppressed,

infected and treated with itraconazole; (SMT/TMP), immunosuppressed, infected and treated

with sulfamethoxazole/trimethoprim; Itra+P10, immunosuppressed, infected, immunized

with P10 and treated with itraconazole; Sulfa+P10, immunosuppressed, infected, immunized

with P10 and treated with SMT/TMP.

Cellular proliferation assay. Spleen cells were collected from mice belonging to the

different groups to assess cellular proliferation to P10 stimulation. Spleen cell were collected,

dispersed manually and washed in RPMI 1640 (Cultilab, Brazil) supplemented with 20mM

NaHCO3, 10mM HEPES, 100 U/ml of penicillin, 100 µg/ml of streptomycin, 2 mM L-

glutamine, 50 μM β-mercaptoethanol, 5mM sodium pyruvate, 100mM non-essential amino

acids (Sigma Chemical Co., St. Louis, MO), and 10% fetal bovine serum (FBS). Cells were

washed twice in FBS-free RPMI, counted, added to 96-well plates at a cell density of 4×105

cells/well at a final volume of 200 µl/well. Subsequently, the splenocytes were stimulated

with P10 peptide for 144 h at 37 ºC in a humidified 5% CO2 incubator. As positive control,

splenocytes from mice of the sham group were stimulated with ConA (4µg/ml, Sigma

Chemical Co., St. Louis, MO). Naïve splenocytes from sham group mice were used as a

negative control. To determine proliferation, MTT (1mg/ml, Thiazolyl Blue Tetrazolium

Bromide – Sigma, St Louis) was added to each well during the final 4 h of culture. The

reaction was stopped with 100 µl/well of isopropanol – HCl 0,04N, and plates were read in a

ELISA reader (Titertek Multiskan EIA reader) at 590 nm wavelength. Data were expressed as

the means and standard deviations (SD) of triplicate cultures.

Cytokine analysis. Cytokine profiles were determined in the supernatants of the splenocyte

cultures obtained in the cellular proliferation assay. Interleukin-4 (IL-4), interleukin-12 (IL-

12), and interferon-gamma (IFN-γ) were measured using ELISA kits (BD Biosciences, San

Diego, CA) and interleukin-1β (IL-1β) was measured using an ELISA kit by eBiosciences,

Inc. (San Diego, CA). The limit of detections for the assays were 7.8 pg/ml for IL-4, 31.25

pg/ml for IFN-, 62.5 pg/ml for IL-12p40, and 8 pg/ml for IL-1β, as previously determined by

the manufacturer.

Production of nitric oxide. Supernatants of the splenocyte cultures obtained in the cellular

proliferation assay were used to determinate the production of nitric oxide (NO), in a NO

chemiluminescence analyzer (NOATM280, Sievers Inc., USA). A calibration curve was

determined using sodium nitrate standards. Using the NOATM280 analyzer, nitrate was

reduced to NO with vanadium (III) at 90 ºC, and the NO formed was detected by gaseous

phase chemiluminescence after reaction with ozone.

Immunohistochemical analysis. Lung tissue samples from infected mice were submerged in

liquid nitrogen then stored at -80 °C until analysis. The frozen sections were cut on a cryostat

(Leica CM1850) and sections of 5 micrometers were applied to poly-L-lysine microscope

slides (Star Frost) and fixed with acetone to perform immunohistochemical analyses. After

wasingh with buffer, endogenous peroxidase was blocked with a 3% solution of hydrogen

peroxide (30%) for 5 minutes. Nonspecific protein binding was blocked with NORMAL

SERUM (Vector Laboratories Vectastain ABC Kit) and BSA 2% (Bovine Serum Albumin,

pH 7.4; Sigma Chemical Co., St. Louis, MO) was used to block endogenous biotin. Slides

were separately incubated for an hour with a dilution of 1:50 (BSA 1% ⁄ Tween 20) with rat

polyclonal antibody anti-mouse CD11b, Ly-6G/Ly-6C and L3T4 (BD PharmigenTM

San

Diego, CA). Biotinylated goat anti-rat IgG (1:500) (Vector Laboratories, Burlingame, CA,

USA) was used to bind rat polyclonal antibody anti-mouse CD11b, Ly-6G/Ly-6C and L3T4,

this was applied for 1 h at room temperature, followed by the addition of streptavidin-

peroxidase (1:50) (Vector Laboratories, Burlingame, CA, USA) for an hour at room

temperature. Chromogen 3,3¢ diaminobenzidine tetrahydrocloride (DAB; Sigma-Aldrich, St.

Louis, MO, USA) was used to localize peroxidase in tissue sections. Finally, the slides were

counterstained with Mayer’s haematoxylin and examined using a light microscope (Nikon

Eclipse E200, Japan).

Fibrosis detection. Lung tissues from infected mice were fixed in 10% buffered formalin, and

then embedded in paraffin for sectioning. Tissue sections were stained with Gomori’s silver

reticulin–stain to assess the changes occurring in the organization of reticulin fibers (collagen

III), and Masson’s trichrome–stained preparations were used to identify collagen I type fibers.

The slides were evaluated under light microscopy.

Survival curve. Six groups of mice (with 6 animals each) pre-treated with DEX were infected

as described. (1) the control group was only infected and get PBS daily (2) group infected and

immunized with P10 (The first immunization was subcutaneous with CFA and subsequently

peptide immunization was i.p. with IFA) (3) infected and treated with ITC, (4) infected and

treated with SMT/TMP, (5) a group infected, immunized with P10 and also treated with ITC

and (6) a group infected, immunized with P10 and also treated with SMT/TMP. Deaths were

registered daily for a 200 day period and the results were statistically analyzed.

Statistical analysis. Statistics were performed using GraphPad Prism5 software (San Diego,

CA). The results were expressed as the means the standard deviations (SD) of the indicated

numbers of animals or experiments. The nonparametric Tukey’s honestly significant

difference test was employed. P values of ≤ 0.05 indicated statistical significance. In the case

of the survival curve, we performed the Log-rank (Mantel-Cox) Test with P values of ≤

0.0001 indicating statistical significance.

RESULTS

Splenocytes from immunosuppressed mice immunized with P10 peptide develop a

lymphoproliferative response when stimulated with the P10 in vitro.

Splenocytes from untreated uninfected and infected mice produced greater

proliferative responses than uninfected or infected mice treated with DEX (Fig. 1). Treatment

of immunosuppressed and infected animals with CFA/IFA or antifungal drugs alone did not

alter the responsiveness of the spleen cells. In contrast, the splenocytes of immunosuppressed

mice that were immunized with P10 peptide produced high levels of cellular proliferation in

comparison to unimmunized infected immunosuppressed mice. Interestingly, the addition of

antifungal therapy to P10 immunization did not further increase splenocyte responsiveness.

These results indicate that P10 alone is a strong proliferation-inducing immunostimulator of

splenocytes in of immunosuppressed mice, and the response is neither impeded or futher

stimulated by the inclusion of antifungal drugs.

P10 peptide induces proinflammatory cytokine production.

Previous studies with corticosteroids suggested that DEX directly inhibit cytokine

production in T cells (2). We showed whether supernatants of spleen cells cultures had high

levels of inflammatory cytokines in mice subjected to DEX and immunized with P10. DEX

treated mice infected with Pb18 and immunized with P10 produced increased amounts of IL-

12, IFN-, and IL-1β compared to controls (Fig. 2). Notably, immunosuppressed and infected

mice treated with IFA/CFA also had an increase of IFN- that was similar to that achieved

with P10. Cytokine profiles in the mice treated with P10 and antifungal drugs were similar to

that achieved with P10 alone. Mice immunosuppressed with DEX, infected with Pb18 and

immunized with P10 showed similar levels of secretion of IL-4 compared to controls (Fig. 2).

Nitric Oxide production.

Since previous studies indicated that corticosteroids such as DEX inhibit nitric oxide

production in macrophages (14), we measured the concentrations of NO in supernatants from

P10 stimulated spleen cell cultures. Significant concentrations of NO were detected in all

groups immunized with P10 compared to controls (Fig. 3). Notably, treatment with either ITC

or SMT/TMP produced results similar to that achieved with P10 alone. Combination

treatments with P10 and antifungal drugs did not further increase NO levels.

Immunosuppressed, infected mice treated with CFA/IFA also displayed an increase in NO

production, albeit significantly less than P10 or antifungal treated animals. The

immunocompromised uninfected or infected but untreated mice had lower levels of NO than

the competent controls, confirming the suppressive effects of DEX on macrophage NO

production.

Phenotypic characterization of CD11b+, Ly-6G/Ly-6C

+ and L3T4

+ cells in lungs from

immunosuppressed BALB/c mice.

We determined the phenotype of CD11b+, Ly-6G/Ly-6C

+ and L3T4

+ cells in the

lesions obtained from immunosuppressed BALB/c mice 60 days after infection with Pb18

(Fig. 4). We observed a significant increase (p < 0.05) in the number of cells CD11b+ in the

pulmonary tissue of animals immunized with P10 with dense clustering of these cells around

rare yeast cells. In contrast, the tissue sections from infected and non-immunized animals had

few CD11b+ cells that were not tightly associated with dispersed yeast cells. These data

strongly indicated that macrophages accumulated in P. brasiliensis induced lesions of

imunosuppressed mice that were immunized with P10 peptide. Within small compact

granulomas of animals immunized with P10, we also found a significant increase (p < 0.05) in

the number Ly-6G/Ly-6C+ cells, which were in close proximity to the rare yeast cells. The

L3T4+ receptor is expressed in a subpopulation of mature T lymphocytes, as MHC class II-

restricted T cells, including most T helper cells that exert an important role in host defense

against fungi. In our experiment, we noted a significant increase (p < 0.05) in the number of

L3T4+ cells in the pulmonary tissue of mice immunized with P10 compared with infected

animals non-immunized (Fig. 4). In the P10 immunized mice, the L3T4+ cells were again

clustered around rare yeast cells.

Immunization with P10 minimizes production of fibrosis in the lung tissue.

We analyzed the quantity and appearance of reticulin and collagen fibers in lungs from

immunosuppressed BALB/c mice 60 days after infection with Pb18 (Fig. 5). In mice treated

only with DEX (controls), the architecture of the lung tissues was significantly disrupted with

large aggregates of yeast cells as well as single fungal cells present diffusely. Moreover, we

observed enough fibers of collagen and reticulin in the pulmonary tissue of these animals

(Fig. 5 A and C). DEX treated animals that received immunization with P10 displayed a

conserved pulmonary tissue architecture with a few compacted granulomas and rare yeast

cells. In contrast with the dense fibrosis in the controls, the lungs of the P10 immunized

animals had significantly less collagen and reticulin (Fig. 5 B and D).

P10 immunization increases survival rates of immunosuppressed mice.

The survival rates of DEX treated, anergic BALB/c mice infected with Pb18 were

significantly impacted by P10 and antifungal thereapy (Fig. 6). In untreated controls, 100%

mortality occurred within the first eighty days post-infection. Animals treated with 10 mg/kg

of itraconazole or 15-3 mg/kg of sulfamethoxazole/trimethoprim, had significantly increased

survival rates of 40% to 50%, respectively. Mice immunized with P10 peptide presented a

60% of survival rate, which was not significantly different from antifungals alone. However,

there were no deaths in mice that were immunized with P10 and treated with either antifungal

drug over a period of two hundred days post-infection. These results confirmed the protective

capacity of immunization with the P10 peptide associated with chemotherapy.

DISCUSSION

Our previous results demonstrate that immunization with P10 peptide

(QTLIAIHTLAIRYAN), led to an additive effect when the immunization was associated with

different drug treatments, inducing a decrease in fungal burden and preventing its spread to

other anatomical sites in the experimental model of paracoccidioidomycosis (16). In this study

we showed the ability of P10 peptide to stimulate and induce specific immune response

against P. brasiliensis in anergic animals infected with the virulent isolate Pb18. P10 has

become a strong candidate vaccine, leading to a predominant Th1-type immune response.

This type of response is the main against P. brasiliensis infection characterized by increased

secretion of IFN-γ, which stimulates the granuloma formation that may contain pathogenic

yeasts (5). IFN-γ is capable to activate lung macrophages, which increases its fungicidal

activity and, therefore, are involved in the resistance to infection by P. brasiliensis. Their

absence contributes to determine the susceptibility to infection (5, 30). Defense against fungal

infections requires a good interaction between the innate and adaptive immune responses.

This report adds new data on the induction of immune responses in mice vaccinated with P10

and shows that, under experimental conditions, this peptide vaccine represents a promising

therapeutic approach for the control of paracoccidioidomycosis.

Cellular immunity is essential in host defense against fungal infection in humans and

in experimental models (25). Immunization with P10 restored the ability of

lymphoproliferation of immunosuppressed animals. Spleen cells from anergic animals

infected with Pb18 and immunized with P10, when stimulated with P10 peptide in vitro

showed a significant higher lymphoproliferation than the splenocytes of non-immunized

animals. Therefore, we think that this increase in lymphoproliferation in vaccinated animals

helps to prevent the fungal spread to other organs. However, in cell culture supernatant of

splenocytes used in the lymphoproliferation assay, we observed an increased secretion of

proinflammatory cytokines such as IL-12, TNF-α, IFN-γ and IL-1β. Previous studies have

demonstrated the important role of these cytokines, especially IFN-γ, which plays an

important role in the protection and resistance to PCM (7). This result tells us that

immunosuppressed animals, when immunized with P10 peptide have a predominant cellular

response when compared to non-immunized animals, thus leading to a reduction in fungal

burden and fall in the spread of the fungus to other organs.

Macrophages are important producers of nitric oxide. Previous studies have shown

that nitric oxide is involved in the control of P. brasiliensis as mycelium or yeast forms (12).

Anergic animals infected with P. brasiliensis and then immunized with P10 showed an

increased production of nitric oxide in the splenocyte culture supernatants. This increase can

be directly or indirectly related to the increase in CD11b + cells.

Through immunohistochemistry, our results demonstrate a significant increase in the

number of CD11b+, Ly-6G

+/Ly-6C

+ and L3T4

+ cells in the lung tissue of immunosuppressed

animals that were previously immunized with P10. The CD11b receptor may be expressed by

macrophages, dendritic cells, natural killer, microglia, and B-1 cells, (1). Dendritic cells and

macrofages are part of the first line of defense against P. brasiliensis (28). Neutrophils and

monocytes are Ly-6G+/ Ly-6C

+ cells that may be involved in the first host response against

infection by P. brasiliensis (19). T lymphocytes (MHC class II-restricted T cell, including

most T helper cells) and the NKT cells are L3T4+ cell type, important in the cellular response,

the major mechanism of host defense in PCM.

It is important to have an increased inflammatory response to contain the infection and

the spread of the fungus in the host, but it is also important to avoid the deleterious effects of

an exacerbated immune response. Pulmonary fibrosis is a severe and progressive sequel of

PCM. Development of pulmonary fibrosis is probably attributed to a prolonged inflammatory

stimulus which is common in PCM (18). Animals immunized with P10 showed a decrease in

pulmonary fibrosis. A likely explanation is the consequent reduction in fungal burden in the

lungs of immunized animals, which led to a decrease in stimulation and consequent reduction

of pulmonary fibrosis.

In addition, we detected increased levels of cytokines such as IFN-γ, TNF-α, IL-1β

and IL-18 in the lungs (data not shown), indicating a restoration of cellular immunity of

anergic animals immunized with P10. In paracoccidioidomycosis TNF-α is directly related to

the formation of granulomas (15) and with IL-12 and IL-18, potentiates the fungicidal activity

of macrophages.

The 100% of the animals immunosuppressed and infected with the virulent isolate

Pb18, died within 80 days after infection. Animals immunized with P10 showed a survival of

50% and there was a 40% survival in those animals treated only with drugs. Mice that were

treated with the drugs sulfamethoxazole/trimethoprim and itraconazole, associated with the

immunization with P10 showed 100% survival for a period of 200 days.

Currently, treatment of PCM is related to the severity of the disease, type of drug

utilized and the time of use. The toxicity of drugs, combined with reports of relapses due to

the resistance of the fungus has been observed (26). This fact makes important to search for

new therapeutic adjuvants in order to decrease the duration of treatment and relapses,

preventing the spread of the fungus to other anatomical sites.

In conclusion we demonstrated that the peptide P10 restores specific immunity

against P. brasiliensis even in animals subjected to immunosuppressive treatment with

synthetic corticosteroids (DEX) in an experimental model of PCM. Use of preventive or

therapeutic vaccines in infectious diseases has expanded and requires the selection of

immunogenic components that may lead to the protection of the host. Efficiency of

immunization with the P10 peptide, as well as the additive effect of the treatment associate

with conventional medications, open new perspectives and makes the P10 an important

vaccine candidate to be tested in human paracoccidioidomycosis.

ACKNOWLEDGMENTS

This work was supported by grants 07/58750-0, 2011/17267-4 from Fundação de Amparo à

Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq).

We acknowledge the valuable technical assistance of Laboratory the immunohistochemistry

of Department of Anatomy the University of São Paulo and Carlos da Silva at the technical

assistance of animal facilities of Department of Microbiology the University of São Paulo.

CONFLICTS OF INTEREST

None to declare.

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27. Shikanai-Yasuda MA, et al. 1995. Paracoccidioidomycosis in a renal transplant

recipient. J. Med. Vet. Mycol. 33:411-414.

28. Silvana dos Santos S, Ferreira KS, Almeida SR. 2011. Paracoccidioides

brasiliensis-Induced Migration of Dendritic Cells and Subsequent T-Cell Activation in

the Lung- Draining Lymph Nodes. PLoS ONE 6(5): e19690.

29. Singer-Vermes LM, Caldeira CB, Burger E, Calich VLG. 1993. Experimental

murine paracoccidioidomycosis: relationship among the dissemination of the

infection, humoral and cellular immune responses. Clin. Exp. Immunol. 94:75-79.

30. Souto JT, Figueiredo F, Furlanetto A, Pfeffer K, Rossi MA, Silva JS. 2000.

Interferon-gamma and tumor necrosis factor-alpha determine resistance to

Paracoccidioides brasiliensis infection in mice. Am. J. Pathol. 156:1811-1820.

31. Taborda CP, Juliano MA, Puccia R, Franco M, Travassos LR. 1998. Mapping of

the T-cell epitope in the major 43-kilodalton glycoprotein of Paracoccidioides

brasiliensis which induces a Th-1 response protective against fungal infection in

BALB/c mice. Infect. Immun. 66:786–793.

32. Vicentini AP, et al. 1994. Binding of Paracoccidioides brasiliensis to laminin

through surface glycoprotein gp43 leads to enhancement of fungal pathogenesis.

Infect. Immun. 62:1465–1469.

FIGURES

Non

-stim

ulat

ed

Con

A

Sham

Infe

cted

Sham

Infe

cted

IFA/C

FAIT

C

SMT/T

MP

P10

P10+IT

C.

P10+SM

T/TM

P

0.0

0.1

0.2

0.3

0.4

0.5

Dex

non-Dex

D.O

. 590

* * *

Figure 1: Immunosuppressed mice immunized with P10 peptide develop a

lymphoproliferative response of splenocytes when stimulated with P10 in vitro.

Splenocytes were isolated from experimental groups after 60 days i.t. infection with 3x105

yeast of P. brasiliensis. The splenocytes were stimulated with P10 peptide in PRMI for

144 hours. Splenocytes from the unifected mice that didn’t receive dexamethasone were

also incubated in RPMI alone (negative control) or with ConA (positive control, 4µg/ml).

* P <0.05significance relative to the control group.

Figure 2: Detection of cytokines in supernatants of spleen cells cultures of

immunosuppressed BALB/c mice after 60 days i.t. infection with 3x105 yeast of P.

brasiliensis. Asterisks indicate statistically significant differences between results detected

in mice immunized with P10 and mice that received only PBS. * P ≤ 0.05, ** P ≤ 0.01.

Sham

Infe

cted

Sham

Infe

cted

IFA/C

FAIT

C

SMT/T

MP

P10

ITC+P10

SMT/T

MP+P10

0

5

10

15

20

25

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***

non-DEX

DEX

NO

(uM

)

Figure 3: Nitric oxide measurements in spleen cells culture of immunosuppressed BALB/c

mice after 60 days i.t. infection with 3x105 yeast of P. brasiliensis. Splenocytes were cultured

in RPMI medium in the presence of P10 peptide for 144 hours. NO levels were detected using

a chemiluminescence analyzer (NOATM280, Sievers Inc., USA). * P ≤ 0.05, *** P ≤ 0.001

relative to the infected group treated with Dex. Error bars denote SD.

A (Control) CD11b

B (Control)

C (Control)

Ly-6G/Ly-6C

L3T4

Non-immunized Immunized with P10

Figure 4: CD11b+, Ly-6G/Ly-6C+ and L3T4+ cells in the lung tissues from

immunosuppressed and infected BALB/c mice. Tissue sections were obtained 60 days

after infection with 3x105 yeast cells of P. brasiliensis. (A) CD11b+ cells in lung

tissue of control group and immunized mice with P10. (B) Ly-6G/Ly-6C+ cells in

lung tissue of control group and immunized mice with P10. (C) L3T4+ cells in lung

tissue of control group and immunized mice with P10. Diaminobenzidine (DAB) was

used as the peroxidase substrate to generate a brown-staining signal and the sections

were counterstained with Mayer hematoxylin. Magnification, X 200.

Figure 5: Evaluation of pulmonary fibrosis in the lungs of immunosuppressed BALB/c

mice infected with 3 x 105

yeast cells the P. brasiliensis 60 days post-infection. (A, C)

Untreated group. (B, D) Immunized with P10 peptide group. (A, B) Masson's Trichrome

staining to detect the fibers of collagen type I and (C, D) Gomori’s silver reticulin staining

to detect collagen type III fibers. Magnification 100X.

0 50 100 150 2000

20

40

60

80

100Control PBS

ITC.

SMT/TMP.

P10

P10+SMT/TMP

P10+ITC.

Days

Su

rviv

al

(%)

Figure 6: Survival curve of immunosuppressed BALB/c mice i.t. infected with 3x105

yeast cells of P. brasiliensis. (●) Control group (PBS). (■) Treated with ITC (10

mg/kg); (▲) Treated with SMT/TMP (15-3 mg/kg); (▼) Immunized with P10; (♦)

Immunized with P10 and treated with SMT/TMP (15-3 mg/kg); (□) Immunized with

P10 and treated with ITC (10 mg/kg); the results are representative of two independent

experiments. p ≤ 0.0001 between different groups.

Artigo 2

Muñoz J. E

162

Artigo 2

MARQUES, A.; DASILVA, M.; JULIANO, M.; MUÑOZ, J. E.; TRAVASSOS, L. R.; TABORDA, C. P. Additive effect of P10 immunization and chemotherapy in anergic mice challenged intratracheally with virulent yeasts of Paracoccidioides brasiliensis. Microbes and Infection, p. 1, 2008.

Original article

Additive effect of P10 immunization and chemotherapy in anergicmice challenged intratracheally with virulent yeasts

of Paracoccidioides brasiliensis

Alexandre F. Marques a, Marcelo B. da Silva a, Maria A.P. Juliano b, Julian E. Munh~oz a,Luiz R. Travassos c, Carlos P. Taborda a,*

a Institute of Biomedical Sciences, Department of Microbiology, University of S~ao Paulo, S~ao Paulo, SP, Brazilb Department of Biophysics, Federal University of S~ao Paulo, S~ao Paulo, SP, Brazil

c Department Microbiology, Immunology and Parasitology, Federal University of S~ao Paulo, S~ao Paulo, SP, Brazil

Received 13 May 2008; accepted 8 July 2008

Available online 24 July 2008

Abstract

Paracoccidioidomycosis is a systemic granulomatous disease manifested in the acute/subacute or chronic forms. The anergic cases of theacute/subacute form are most severe, leading to death threatening conditions. Drug treatment is required to control the disease but the response inanergic patients is generally poor. A 15-mer peptide from the major diagnostic antigen gp43, named P10, induces a T-CD4þ helper-1 immuneresponse in mice of different haplotypes and protects against intratracheal challenge with virulent P. brasiliensis. Presently, P10 immunizationand chemotherapy were associated in an attempt to improve antifungal treatment in Balb/c mice made anergic by adding dexamethasone to thedrinking water. The combined drug/peptide treatment significantly reduced the lung CFUs in infected anergic mice, largely preserved lungalveolar structure and prevented fungal dissemination to liver and spleen. Results recommend that a P10-based vaccine should be associated tochemotherapy for improved treatment of paracoccidioidomycosis aiming especially at anergic cases.� 2008 Elsevier Masson SAS. All rights reserved.

Keywords: P. brasiliensis; Anergy; Dexamethasone; Chemotherapy; Immunization; P10; gp43

1. Introduction

Paracoccidioidomycosis (PCM) is a systemic granulomatousdisease caused by Paracoccidioides brasiliensis, a thermallydimorphic fungus widespread in Latin America mainlyaffecting rural workers. An estimate on the incidence of PCM inLatin America showed that approximately 10 million peoplemay be infected and 2% of them may develop the disease [1].The incidence may increase in recent areas of deforestation anddevelopment with the practice of soil churning, as well as inimmunocompromised individuals [2]. In the 1980e1995 period

there occurred 3181 lethal cases only in Brazil, but this isprobably underestimated, since the disease is not classified as ofmandatory notification by the governmental health authority [3].

The disease can exhibit two forms, acute/subacute andchronic. The former equally affects both genders and primarilyinvolves the reticuloendothelial/lymphatic system. The chronicform affects mainly adult males with predominant pulmonaryand/or mucocutaneous involvement [4]. Activation of animmune cellular response is the primary effective mechanism ofcontrol of experimental and human PCM and a correlation hasbeen found between the severity of the disease and impaireddelayed-type hypersensitivity (DTH) response [5].

Antifungal chemotherapy is required to control the disease.Initial treatment with sulfonamides, amphotericin B, or azolesmay last from 2 to 6 months. Extended periods of treatmentare often necessary, up to 2 or more years [6]. Significantfrequency of relapsing disease is observed in patients

* Corresponding author. Instituto de Ciencias Biomedicas II, Departamento

de Microbiologia, Universidade de S~ao Paulo, Av. Prof. Lineu Prestes, 1374,

2� andar, S~ao Paulo, SP 05508-900, Brazil. Tel.: þ55 11 3091 7345; fax: þ55

11 3091 7354.

E-mail address: [email protected] (C.P. Taborda).

1286-4579/$ - see front matter � 2008 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.micinf.2008.07.027

Microbes and Infection 10 (2008) 1251e1258www.elsevier.com/locate/micinf

mailto:[email protected]

http://www.elsevier.com/locate/micinf

submitted to short periods of treatment (reviewed in ref. [7]).Anergic conditions are death threatening due to poor responseto chemotherapy when the immune system collapses.

The gp43 is the major diagnostic antigen of P. brasiliensis. Anti-bodies to the glycoprotein can be used in the follow-up of patientsusing avariety of serological tests [8,9]. Also, it was demonstrated thatgp43 can elicit delayed hypersensitivity reactions in guinea pigs [10]and humans [11], implying the presence of T-CD4þ reacting epitopes.

The gp43 gene was cloned and sequenced encoding a poly-peptide of 416 amino acids (Mr 45,947) with 56e58% similaritywith exo-1,3-b-D-glucanases from Saccharomyces cerevisiaeand Candida albicans [12]. The mature protein has a single highmannose N-glycosylated site [13]. Investigation of the gp43gene polymorphism showed that the P10 encoding sequence ishighly conserved with rare mutations in this region [14].

The H-2d-restricted T-cell epitope was mapped to a 15-merpeptide called P10. An inner core of P10 formed by HTLAIR isthe essential domain of the epitope inducing proliferation oflymph node cells from mice sensitized to the gp43 or infectedwith P. brasiliensis. Lymphoproliferation induced by either P10or gp43 involved CD4þT-helper lymphocytes producing IL-2and IFN-g. Immunization of mice with both gp43 and P10 in thepresence of complete Freund’s adjuvant (CFA) caused a 200-fold decrease in CFU from the lung of mice challenged intra-tracheally with virulent yeasts of P. brasiliensis [15].

Recently we have shown that P10 immunization associated tochemotherapy improved antifungal treatment and preventedrelapses. In all cases the combined immunochemotherapyshowed an additive protective effect clearly superior to drug andP10-vaccine administered alone. P10-vaccination couldsuccessfully reverse the relapsing infection observed in sul-phamethoxazole/trimethoprim treated mice. Generally, drugtreatment favored a Th-2 response producing IL-4 and IL-10,whereas immunization with P10 stimulated a pro-inflammatoryTh-1 response with increased IL-12 and IFN-g. In successfullytreated mice both Th-1 and Th-2 cytokines were present. Thecombined drug/vaccine treatment significantly reduced the lungCFUs, largely preserved lung alveolar structure and preventedfungal dissemination to liver and spleen [16].

In the present work we investigated the effectiveness of P10in an experimental model that aimed at mimetizing the anergicstate, common in patients with the acute and sub acute forms.In general, we studied the behavior of immunosuppressedanimals, infected and treated with itraconazole or sulfame-thoxazole and trimethoprim, associated or not with theimmunization with P10. This novel treatment was shown to bequite effective in mice intratracheally infected with this fungusand with the perspective of potentially reducing the time oftreatment in human patients, prevent relapsing disease andeventually reverse life threatening anergic cases.

2. Materials and methods

2.1. Animals

Balb/c mice (6- to 8-week-old males) were bred at Univer-sity of S~ao Paulo animal facility under specific-pathogen-free

conditions. Procedures involving animals and their care wereconducted in conformity with the local Ethics Committee andinternational recommendations.

2.2. Fungal strain

Virulent P. brasiliensis Pb18 yeast cells were maintained byweekly passage on solid Sabouraud medium at 37 �C and wereused after 7e10 days of growth. Before experimental infec-tion, the cultures were grown in modified McVeigheMortonmedium (MMcM) at 37 �C for 5- to 7-days [17]. The fungalcells were washed in phosphate-buffered saline (PBS, pH 7.2)and counted in a hemocytometer. The viability of fungalsuspensions determined by staining with trypan blue (Sigma,St Louis, MO) was always higher than 90%. The virulence ofisolate Pb18 was checked in each experiment by infectingintratraqueally Balb/c mice and recovering the yeast cells fromthe organs of mice.

2.3. Immunosuppression of mice

To examine the effects of immunosuppression in miceintratracheally infected with P. brasiliensis, dexamethasonephosphate (Sigma, St Louis, MO) was added to the drinkingwater of infected animals. Assuming an average water intakeof 5 ml per day for 30 days, the daily dexamethasone phos-phate dose was calculated as 0.15 mg kg�1.

2.4. Delayed-type hypersensitivity (DTH) reaction

To verify the effect of dexamethasone administration theDTH reactions against Pb-18 antigen were measured as wellthe evolution of the infectious process was evaluated. The lefthind footpad was injected subcutaneously with 25 ml of FavaNetto’s antigen every 3 days [18] and variations in size(diameters) were measured after 24 and 48 h.

2.5. Peptide synthesis and purification

Peptide synthesis and purification was carried out at theDepartment of Biophysics, UNIFESP as described previously[15]. HPLC analysis showed that the synthetic P10 in theamidated form was 90% pure.

2.6. Immunization of mice

Anergic Balb/c mice (6- to 8-week old males) were weeklyimmunized for 4 weeks with 20 mg of P10 each. The firstimmunization was subcutaneous with complete Freund’sadjuvant (CFA) and subsequently peptide immunization wasi.p. with incomplete Freund’s adjuvant (IFA). Control micewere injected with CFA alone.

2.7. Intratracheal infection of Balb/c mice

Balb/c mice were inoculated intratracheally with 3 � 105

yeast cells of virulent P. brasiliensis Pb18, grown on

1252 A.F. Marques et al. / Microbes and Infection 10 (2008) 1251e1258

Sabouraud agar and suspended in sterile saline (0.85% NaCl),per animal. A maximum volume of 50 ml was inoculated permouse. Briefly, mice were anesthetized i.p. with 200 mL ofa solution containing 80 mg/kg of ketamine and 10 mg/kg ofxylazine (both from Uni~ao Quımica Farmaceutica, Brazil);after approximately 10 min, their necks were hyper-extended,and the trachea was exposed at the level of the thyroid andinjected with 3 � 105 yeast cells in PBS using a 26-gaugeneedle. The incisions were sutured with 5-0 silk.

2.8. Fungal burden in organs of infected mice

Mice were sacrificed 45 days after i.t. infection and thefungal burden was measured by colony forming units (CFU).Sections of the lung, liver and spleen were removed, weighed,homogenized and then washed three times with PBS. The cor-responding pellets were re-suspended and homogenized each in1 ml of PBS. A 100 ml-sample of this suspension was plated onsolid braineheart infusion (BHI) medium supplemented with4% fetal calf serum (Gibco, NY, USA), 5% spent P. brasiliensisisolate-192 culture supernatant, streptomycin/penicillin 10 IU/ml (Cultilab, Brazil) and cycloheximide 500 mg/ml (Sigma, StLouis, MO). Petri dishes were incubated at 37 �C for at least10 days, and colonies were counted (1 colony ¼ 1 CFU).

2.9. Chemotherapy of anergic infected mice

The anergic state was obtained 20 days after the adminis-tration of dexamethasone. Animals were then infected anddrug treatment started 15 days after i.t. infection. The treat-ment was held for 30 days during which groups of micereceived doses every 24 h of itraconazole (10 mg/kg), or sul-famethoxazole/trimethoprim (15 mg and 3 mg/kg). All drugadministrations were i.p.

2.10. Groups studied

Eight groups of mice (with 10 animals each) were used.Three controls were included: sham, a group that was neitherinfected nor treated with dexamethasone, antifungals or withP10; a second control group was infected with Pb 18, receiveddexamethasone in the drinking water but was not furthertreated with either an antifungal drug or P10; a third controlgroup included mice infected but not treated with dexameth-asone, antifungal drugs or P10. Five anergic groups included:(a) a group infected and immunized with P10; (b) two groupsof mice infected and treated with either itraconazole or sul-phamethoxazole/trimethoprim; and (c) two groups treated withantifungal drugs and also immunized with P10. All five groups(anergic) received dexamethasone in the drinking water. Thewhole experiment was repeated twice with reproducibleresults.

2.11. Cytokine analysis

Mice were sacrificed 45 days after infection and sections ofthe lung (right and left alternatively), liver and spleen were

homogenized in 2 ml of PBS in the presence of proteaseinhibitors (Complete Mini; Boehringer Mannheim, Indian-apolis, IN). The homogenates were centrifuged, and thesupernatants frozen at �80 �C until tested. The supernatantswere assayed for IL-2, IL-4, IL-10, IL-12, and IFN-g usingELISA kits (BD PharMingen, San Diego, CA). The detectionlimits of such assays were as follows: 3.1 pg/ml for IL-2;7.8 pg/ml for IL-4; 31.25 pg/ml for IL-10 and IFN-g; and62.5 pg/ml for IL-12p40, as previously determined by themanufacturer.

2.12. Antibody response to gp43

The antibody response to gp43 was determined 45 daysafter infection. Microtiter plates sensitized with 500 ng ofpurified gp43 were incubated at 37 �C for 1 h with 100 ml ofmouse serum, serially diluted starting at 1:200. The reactionwas quantified with goat anti-mouse Ig for 1 h at 37 �C, fol-lowed by donkey anti-goat Ig-biotin conjugate and reactionwith streptavidin-peroxidase for 30 min at 37 �C. After addi-tion of the orthophenylenediamine reagent (500 mg/ml), thereaction was read at 492 nm. The antibodies, conjugates, andsubstrate were from Sigma Chemical Co (St Louis, MO).

2.13. Histopathology

The lungs were excised, fixed in 10% buffered formalin,and embedded in paraffin for sectioning. The sections werestained with hematoxylin-eosin or silver nitrate and examinedmicroscopically at 25� magnification (Optiphot-2; Nikon,Tokyo, Japan).

2.14. Statistical analysis

Statistical analysis was done using GraphPad Prism5 soft-ware. The results were expressed as means � SD. The non-parametric Tukey test was used. Unpaired Student’s t-test withWelch’s correction (two-tailed) was used for the comparisonof two groups when the data met the assumptions of the t tests.p < 0.05 indicated statistical significance. Survival experi-ments were compared by log rank analysis (SigmaStat; SPSS,Chigaco, IL).

3. Results

3.1. Determination of the anergic state in Balb/c mice

A group of infected animals were treated with dexameth-asone in the drinking water and every 3 days the animals weresubmitted to a DTH test to determine the time necessary forcomplete anergy. No DTH reaction was observed after 20 daysof treatment with dexamethasone (Fig. 1A). The mortality ingroups of infected mice submitted or not to dexamethasonetreatment was scored up to 100 days after infection. It wasrestricted to the group of animals that received dexamethasonephosphate in the drinking water in the time range of theexperiment (Fig. 1B). Unlike the control group, all animals

1253A.F. Marques et al. / Microbes and Infection 10 (2008) 1251e1258

were killed by day 70. After determining the time necessaryfor anergy induction, all subsequent experimental infectionswere carried out after 20 days of exposure to dexamethasonein the drinking water. Infected anergic mice submitted to P10vaccination and/or antifungal drug treatment were all alive inthe 100-day period of the experiment much like the controlmice untreated with dexamethasone (data not shown).

3.2. Organ CFU from intratracheally infected anergicBalb/c mice immunized with P10 and/or treated withantifungal drugs

Our previous work has shown that P10 could be used asadjuvant in the treatment of established experimental infection[16]. To explore the effects of P10 immunization in anergic Balb/c mice, animals’ immunization started 15 days after infection.Analysis of organ CFUs was done after 45 days of infection. Asignificant reduction of fungi recovered from lung, spleen and

liver of animals (CFU) was obtained in mice immunized withP10. The treatment with itraconazole and sulfamethoxazole/trimethoprim also reduced the fungal burden; a more significantreduction, however, was obtained when itraconazole or sulfa-methoxazole/trimethoprim and P10 immunization werecombined in the treatment of anergic Balb/c mice (Fig. 2).

3.3. Lung histopathology from anergic Balb/c micevaccinated with P10 only, after intratracheal infection

Lungs of infected animals submitted to dexamethasonetreatment showed multiple pulmonary foci of epithelioid gran-ulomatous inflammation, with a loose architecture containinga great number of neutrophils and fungal cells (Fig. 3A). Lungs ofanimals submitted to P10 immunization showed few welldemarcated epithelioid granulomas, compact and with fewfungal cells (Fig. 3B). A similar pattern of lung histopathologywas observed in the tissues of mice treated with itraconazole(Fig. 3C) or sulfamethoxazole/trimethoprim (Fig. 3E). Theassociation of drugs and immunization with P10 resulted in areaswith very few or undetectable yeast cells (Fig. 3D,F). Clearly, inthis case, P10-immunization was an essential adjuvant therapythat reduced the infection. Few untreated animals showed, duringthe experiment, brain dissemination of fungal elements that wasabsent in the P10 immunized mice (data not shown).

3.4. Cytokine detection in the lungs of anergic infectedBalb/c mice immunized with P10 and/or treated withantifungal drugs

Previous studies have established that P10 was able to activatea Th-1 immune response with the known pattern of cytokineproduction in vitro and in vivo [15,16]. Presently, lung cytokinelevels were measured in anergic mice infected with P. brasiliensisand treated with itraconazole or sulfamethoxazole/trimethoprimcombined or not with P10 immunization. A sham group wasevaluated for basal levels of cytokines as well as a control group ofinfected-only mice untreated with dexamethasone, antifungaldrugs or peptide as shown in Table 1. The comparative cytokinelevels among infected Balb/c mice treated or not with dexameth-asone were then evaluated. Results showed that infected micetreated with dexamethasone had a much higher concentration ofIL-4 and IL-10 than of IL-12 and IFN-g. Treatment of mice withantifungal drugs led to partial reduction of IL-4 and IL-10 levelswhich were then further reduced with the association of P10immunization and the antifungal drug. P10 immunization as wellas its association with antifungal treatment markedly enhancedIL-12 and IFN-g levels. Thus, although both Th-1 and Th-2 typesof immune response seemed present in all cases, incorporation ofP10 greatly stimulated a Th-1 response with increased productionof IL-12 and IFN-g, clearly associated with protection.

3.5. Antibody titers against gp43 after 45 daysof infection

Before sacrifice for other determinations, blood wascollected from infected animals and the serum antibodies to

Days of infection

0 10 15 20 25

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ot p

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m)

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% S

urvival

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Fig. 1. Delayed-type hypersensitivity and Survival curve of Balb/c mice. (A)

Follow-up of DTH reactions in infected Balb/c mice (B) and infected,

immunosuppressed Balb/c mice (C). Dotted line represents averages of

uninfected Balb/c mice. Error bars denote standard deviations. (B) Survival of

Balb/c mice infected with 3 � 105 yeast cells of P. brasiliensis Pb18 treated

with dexamethasone (0.15 mg/kg) in the drinking water for 30 days (C);

untreated control (B). The experiments were repeated twice with similar

results.


the gp43 major antigen were titrated. Animals treated withdexamethasone and infected, showed after 45 days by ELISAan antibody titer of 1/6400 compared to 1/200 in mice infectedand submitted to immunization with P10. Anergic miceimmunized with P10 and treated with either itraconazole orsulfamethoxazole/trimethoprim also produced fewer specificantibodies than those treated only with antifungal drugs.

4. Discussion

The main clinical forms of PCM are: acute/subacute andchronic. The acute form is more severe and represents 3e5%of all cases of PCM affecting mainly children and teenagersbut eventually also adults up to 35 years old (reviewed in ref.[6]).

Clinical evidence as well as experimental studies haveshown that cell-mediated immunity is the chief mechanism ofhost defense. Acute/subacute forms are more frequentlyassociated with anergic immune response whereas the chronicform is hyperergic. Acute/subacute forms tend to shownecrotizing lesions with abundant fungal cells, impairment ofcell-mediated immunity and high titers of circulating anti-bodies. In contrast, the chronic form is seldom disseminated,and is associated with tuberculoid granulomata and few fungalcells. The cell-mediated immunity is better preserved with lowantibody levels [19,20].

The control of the disease is made by antifungal drugs suchas sulphonamides, amphotericin B and azoles (reviewed in ref.[7]). Itraconazole is a good option to start the treatment butsulphamethoxazole/trimetoprim therapy is still being used inpublic hospitals in Brazil [6].

The use of peptides as therapeutic vaccines in the treatmentof fungal infections as they have being used in cancer [21]could be an adjuvant to chemotherapy potentially able toreduce the toxicity of conventional drugs as well as the time of

treatment, prevent relapsing disease and even help to treatanergic cases [22,23].

The immunoprotective properties of P10 were well deter-mined in a murine model of PCM. Injection of P10 emulsifiedin complete Freund’s adjuvant (CFA) protected mice againstintratracheal challenge with virulent P. brasiliensis isolate.Disease progression was followed by counting the CFUs intissues and by histopathology. The protective effect of P10 isundoubtedly related to the induction of an IFN-g-dependentTh-1 immune response [15]. A P10-derived multiple-antigen-peptide construction (MAP) administered without CFAconferred equal protection in i.t. infected mice [24]. We haveshown then that an additive protective effect was achieved bythe combination of drug treatment and P10 immunization [16].Aiming at defining the role of P10 as an adjuvant immunetreatment in drug treated, infected animals, we presentlyextended this study to determine the capacity of P10 to reversethe anergic state of Balb/c mice induced by dexamethasone.The recognized anergic state was observed after 20 days oftreatment with dexamethasone in the drinking water a condi-tion that caused death of all infected mice after 70 days ofinfection. The use of dexamethasone to induce the anergicstate in mice has been described previously [25].

Anergic animals i.t. infected with virulent P. brasiliensisshowed high CFU in the lungs and also in the liver, spleen andbrain after 45 days of infection. Cytokine determinationshowed increased IL-4 and IL-10 and low levels of IFN-g andIL-12, a pattern that was reversed by P10 immunization andless efficiently by antifungal drugs. Combination of P10 andantifungal drugs promoted the best Th-1 type immuneconversion. Anti-gp43 IgG titers were higher in the dexa-methasone and antifungal treated groups than in the P10-immunized ones. Immunization of anergic Balb/c mice withP10 led to significant reduction of CFU in the lung, spleen andliver. The protective properties of IFN-g in the murine model

Fig. 2. Colony forming units from organs of infected mice. Colony forming units (CFU) from lungs (L), spleen (S) and liver (V) of Balb/c mice infected

intratracheally with 3 � 105 yeast cells of P. brasiliensis Pb18 (non-anergic) or infected and treated with dexamethasone (anergic). Groups of anergic animals were

also submitted to P10 immunization (P10), itraconazole (Itra) or sulfamethoxazole/trimethoprim (SMT/TMP) treatment, or the combined P10 immunization and

chemotherapy (itraconazole þ P10 and SMT/TMP þ P10). Bars represent the CFU means and standard deviations from organs of five to 10 animals in each group.

* significant ( p < 0.05) difference in relation to the group of mice infected and treated only with dexamethasone in drinking water. ** significant ( p < 0.05)

differences between the combined treatment of P10 and antifungal drug, and the individual treatments with each agent.


have been established previously. Survival analysis of micedeficient in IFN-g or IFN-g receptor (but not IFN-a-R or IFN-b-R), and IRF-1 showed 100% mortality 3e4 weeks afterintratracheal challenge with a virulent isolate. Consistent withthe need of IFN-g for effective immunization with P10, non-immunized infected IFN-g-R knock-out mice displayed thesame mortality rate as of P10-immunized KO mice [9].

Histopathology of infected organs confirmed the protectiveeffect of P10, either by significantly reducing the number ofgranulomas or, when associated with antifungal drugs, leadingto fungal elimination but not sterility because of the short timeof the experimental design [26].

The use of P10 as an adjuvant to antifungal treatment is apromising tool in the treatment of experimental

Fig. 3. Histopathology of lungs from intratracheally infected anergic Balb/c mice submitted to immunization with P10 and/or chemotherapy. Additive protective

effect of P10 immunization and antifungal drug treatment is shown after 45 days of infection. (A) Lung section from infected mouse treated only with dexa-

methasone; (B) same as (A) but immunized with P10, showing a single granuloma; (C) lung section from anergic mouse treated with intraconazole; (D) same from

anergic mouse immunized with P10 and treated with itraconazole; (E) lung section form anergic mouse treated with sulfamethoxazole/trimetoprim (SMT/TMT)

and (F) same from anergic mouse immunized with P10 and treated with (SMT/TMT). Hematoxylin eosin staining; magnification, �25.


paracoccidioidomycosis with a role also in the prevention ofrelapses. The combined treatment could be important even incases of clinical resistance to azoles and sulfamethoxazole/trimethoprim [7,16]. Presently, we report on the additiveprotective effect of itraconazole or sulfamethoxazole/trimeth-oprim and P10 immunization in anergic animals. AnergicBalb/c mice treated only with antifungal drugs still showedhigh levels of IL-4 and IL-10 possibly due to increased fungaldestruction and high B-cell stimulation. The combined treat-ment with a P10-vaccine clearly stimulated a protective Th-1response rich in IL-12 and IFN-g. A predominant Th-1immune response simultaneous with a persisting but lessintense Th-2 response seems to represent the adequate balanceleading to protection of infected animals. Most probably T-dependent antibodies are also implicated in the immunopro-tection but with the exception of anti-gp43 antibodies whichmay include protective and non-protective types [27] this wasnot further explored presently.

Several features have been examined for validation of P10 asa candidate vaccine antigen including: (a) the presentation ofP10 by different mouse haplotypes [15]; (b) its conservation innature determined by examining gp43 molecules from differentisolates [28]; (c) its immunogenicity in formulations that do notrequire complete Freund’s adjuvant [24,29]; (d) its presentationby human HLA-DR molecules and that of other promiscuouspeptides derived from the gp43 [30]; and (e) additive effect whenadministered with antifungal drugs [16] and finally, its role in theimmunoprotection of anergic mice infected with P. brasiliensisand submitted to chemotherapy which was the focus of thepresent work. The present data strongly suggest that a P10-basedvaccine may be used to enhance antifungal protection bychemotherapy even in cases of anergy.

Acknowledgements

CPT and LRT were supported by Fundac~ao de Amparo aPesquisa do Estado de S~ao Paulo (Fapesp) grants 07/07588-2

and 06/50634-2, CNPq grant 470636/2007-6. CPT and LRTare research fellows of the CNPq.

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Table 1

Lung cytokine levels after 45 days of infection in anergic Balb/c mice infected with 3 � 105 yeast cells of P. brasiliensis Pb18

Cytokine Cytokine levels (ng/ml of tissue)

Shama Infected onlyb Infected dexamethasonec P10d Itraconazolee Itraconazole and P10f SMT/TMPg SMT/TMP and P10h

IL-2 1.71 � 0.7 2.51 � 0.56 1.98 � 0.94 8.09 � 0.32 3.16 � 1.0 8.07 � 1.98* 3.4 � 1.35 6.51 � 0.56*

IL-4 0.2 � 0.06 3.37 � 0.69 10.9 � 1.86 1.14 � 0.46 4.86 � 0.38 0.68 � 0.19* 5.56 � 0.61 0.62 � 0.12*

IL-10 4.39 � 1.87 12.52 � 1.76 20.16 � 5.24 8.16 � 2.99 8.66 � 2.52 3.64 � 2.04* 13.28 � 2.64 6.53 � 2.16*

IL-12 0.58 � 0.23 2.12 � 0.50 1.52 � 0.25 10.48 � 1.53 2.81 � 0.43 11.6 � 1.38* 2.2 � 0.48 10.05 � 0.63*

IFN-g 0.38 � 0.26 0.31 � 0.11 1.1 � 0.7 4.5 � 0.88 1.18 � 0.30 7.39 � 1.34* 0.41 � 0.22 6.54 � 0.77*

*Significant differences ( p < 0.05) relative to anergic mice submitted to chemotherapy only. Values are means for 10 animals per group with standard deviations.

The whole experiment was repeated twice with reproducible results.a Uninfected, non-immunized and untreated animals (n ¼ 10).b Infected only, untreated with dexamethasone, antifungal drugs or P10.c Infected only, but treated with dexamethasone.d Treated with dexamethasone (anergic), infected and immunized with P10.e Anergic, treated with itraconazole.f Anergic, treated with itraconazole and P10 immunization.g Anergic, treated with sulfamethoxazole/trimetoprim (SMT/TPM).h Anergic, with SMT/TPM and P10 immunization.


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Artigo 3

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Artigo 3

BUISSA-FILHO, R.; PUCCIA, R.; MARQUES, A. F.; PINTO, F. A.; MUÑOZ, J. E.; NOSANCHUK, J. D.; TRAVASSOS, L. R.; TABORDA, C. P. The Monoclonal Antibody against the Major Diagnostic Antigen of Paracoccidioides brasiliensis Mediates Immune Protection in Infected BALB/c Mice Challenged Intratracheally with the Fungus. Infection and Immunity, v. 76, p. 3321-3328, 2008.

INFECTION AND IMMUNITY, July 2008, p. 3321–3328 Vol. 76, No. 70019-9567/08/$08.00�0 doi:10.1128/IAI.00349-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Monoclonal Antibody against the Major Diagnostic Antigen ofParacoccidioides brasiliensis Mediates Immune Protection in

Infected BALB/c Mice Challenged Intratracheallywith the Fungus�

R. Buissa-Filho,1 R. Puccia,2 A. F. Marques,1 F. A. Pinto,1 J. E. Munoz,1J. D. Nosanchuk,3 L. R. Travassos,2 and C. P. Taborda1*

Institute of Biomedical Sciences, Department of Microbiology, University of Sao Paulo, Sao Paulo, SP, Brazil1; Department ofMicrobiology, Immunology and Parasitology, Federal University of Sao Paulo, Sao Paulo, SP, Brazil2; and Departments of

Medicine and Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York3

Received 17 March 2008/Returned for modification 8 April 2008/Accepted 24 April 2008

The protective role of specific antibodies against Paracoccidioides brasiliensis is controversial. In the presentstudy, we analyzed the effects of monoclonal antibodies on the major diagnostic antigen (gp43) using in vitroand in vivo P. brasiliensis infection models. The passive administration of some monoclonal antibodies (MAbs)before and after intratracheal or intravenous infections led to a reduced fungal burden and decreasedpulmonary inflammation. The protection mediated by MAb 3E, the most efficient MAb in the reduction offungal burden, was associated with the enhanced phagocytosis of P. brasiliensis yeast cells by J774.16, MH-S,or primary macrophages. The ingestion of opsonized yeast cells led to an increase in NO production bymacrophages. Passive immunization with MAb 3E induced enhanced levels of gamma interferon in the lungsof infected mice. The reactivity of MAb 3E against a panel of gp43-derived peptides suggested that the sequenceNHVRIPIGWAV contains the binding epitope. The present work shows that some but not all MAbs againstgp43 can reduce the fungal burden and identifies a new peptide candidate for vaccine development.

Paracoccidioides brasiliensis is a thermally dimorphic fungusthat is the etiological agent of paracoccidioidomycosis (PCM),the most prevalent systemic mycosis in Latin America that isendemic in areas of Brazil, Argentina, Colombia, and Vene-zuela. The impact of the disease is demonstrated by Coutinhoet al. (14), who reported that 3,181 lethal cases of PCM oc-curred in the 1980 to 1995 period in Brazil. The acute andsubacute forms of PCM affect both genders and primarilyinvolve the reticuloendothelial/lymphatic system. The chronicform affects mainly adult males with predominant pulmonaryand/or mucocutaneous involvement (20). The activation of theimmune cellular response is the primary effective mechanismto control experimental and human PCM (4, 25). A correlationhas been found between the severity of the disease and animpaired delayed-type hypersensitivity response (30).

Antifungal chemotherapy is required for PCM treatment,though even after prolonged administration, there is no assur-ance of the complete destruction of the fungus. The period oftreatment depends on the drug used and disease severity. Stan-dard drugs include sulfonamides, amphotericin B, and azoles(44).

gp43, first described by Puccia et al. (35), is the major diag-nostic antigen of P. brasiliensis used in a variety of serologicaltests (16, 50). Antibody titers to gp43 have been used to mon-

itor the response to treatment in patients (6). gp43 has alsoproven to be immunodominant in a crude antigenic prepara-tion, eliciting delayed hypersensitivity reactions in guinea pigs(40) and humans (42), which indicated the presence ofT-CD4�-reacting epitopes.

The gp43 gene has been cloned and sequenced (13). Itencodes a polypeptide of 416 amino acids (Mr, 45,947) with aleader peptide of 35 residues; the mature protein has a singlehigh-mannose N-glycosylated chain (1). The H-2d-restrictedT-cell epitope has been mapped to a 15-mer peptide called P10(48). Different 12-mer sequences, all containing the hexapep-tide HTLAIR, induce the proliferation of lymph node cellsfrom mice sensitized to gp43 or infected with P. brasiliensis.Lymphoproliferation induced by either P10 or gp43 involvesCD4� T-helper lymphocytes producing interleukin-2 (IL-2)and gamma interferon (IFN-�). The immunization of micewith either gp43 or P10 in complete Freund’s adjuvant signif-icantly protected them from intratracheal (i.t.) infection withvirulent yeasts of P. brasiliensis. After 3 months of infection,the lung CFU were �200-fold fewer than in the untreatedcontrol mice.

The role of antibody-mediated immunity in host resistanceto P. brasiliensis is less certain (10). In several systems, how-ever, there is considerable evidence that the administration ofmonoclonal antibodies (MAbs) can modify the course of dis-ease in mice infected with fungi such as Cryptococcus neofor-mans (28), Candida albicans (15, 24), Histoplasma capsulatum(31), Pneumocystis spp. (51), Fonsecaea pedrosoi (2), Aspergillusspp. (12), and, more recently, P. brasiliensis (17).

The mechanisms of antibody action in combatting an infec-tious disease include antigen neutralization, cooperative effects

* Corresponding author. Mailing address: Institute of BiomedicalSciences, Department of Microbiology, University of Sao Paulo,Ave. Prof. Lineu Prestes, 1374, 2° andar, Sao Paulo, SP 05508-900,Brazil. Phone: 55-11-3091-7345. Fax: 55-11-3091-7354. E-mail:[email protected].

� Published ahead of print on 5 May 2008.

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with cellular immunity by the enhancement of phagocytosis ormediating-cell cytotoxicity, complement activation, growth in-hibition, adherence, and biofilm or direct antimicrobial effects(reviewed in reference 11). With C. neoformans, it is clear thatantibody-mediated immunity can be decisive for host defense.The mechanisms involved are complex and dependent on sev-eral parameters, such as antibody isotype (26), T-cell function(53), C. neoformans strain (29), antibody quantity (49), expres-sion of inducible NO synthase (39), Fc region, and comple-ment activation (43).

The control of PCM is classically associated with a vigorousTh1 response and granuloma formation. There is evidence,however, that antibody-mediated immunity can contribute toinfection clearance. In the present study, a panel of MAbsagainst gp43 was utilized for evaluating the effect of passiveimmunization in mice intravenously (i.v.) or i.t. infected with avirulent strain of P. brasiliensis.

MATERIALS AND METHODS

Animals. BALB/c mice (6- to 8-week-old males) were bred at the University ofSao Paulo, Sao Paulo, Brazil, animal facility under specific pathogen-free con-ditions. The procedures involving animals and their care were conducted accord-ing to the local ethics committee and international rules.

Fungal strain. Virulent P. brasiliensis Pb18 yeast cells were maintained byweekly passage on solid Sabouraud medium at 37°C and were used after 7 to 10days of growth. Before the experimental infection, the fungus was grown inmodified McVeigh-Morton medium at 37°C for 5 to 7 days (38). The fungal cellswere washed in phosphate-buffered saline (PBS; pH 7.2) and counted in ahemocytometer. The viability of the fungal suspensions, determined by stainingwith Janus B (Merck, Darmstadt, Germany), was always higher than 90%.

MAbs. The MAbs against gp43—19G, 10D, 32H, and 17D (immunoglobulinG2a [IgG2a]) and 21F and 3E (IgG2b)—were previously characterized (36). IgGascites was generated in BALB/c mice given intraperitoneal (i.p.) injections ofMAb hybridomas. IgG MAbs were purified from ascitic fluid by protein A affinitychromatography (Pierce, Rockland, IL) as per the manufacturer’s instructions.Endotoxin (lipopolysaccharide) concentration was �1 ng/ml, measured by theLimulus amebocyte test (BioWhittaker, Walkersville, MD). The antibody con-centration was determined by enzyme-linked immunosorbent assay (ELISA)relative to isotype-matched standards. Irrelevant IgG MAb was used as a controland was provided by Elaine G. Rodrigues, Universidade Federal de Sao Paulo.

Endotoxin-free preparations. Disposable pyrogen-free plasticware and endo-toxin-free water or PBS were used in all experiments. Ascitic fluid and isolatedMAb were further purified in Detoxi-Gel columns (Pierce, Rockland, IL) toremove any endotoxin contamination.

Phagocytosis assay. Peritoneal and alveolar macrophages from BALB/c micewere harvested by washing the abdominal cavities or the lungs and culturingadherent cells in Dulbecco’s modified Eagle’s medium with 10% heat-inactivatedfetal calf serum and 1% nonessential amino acids (Cultilab, Brazil). Otherphagocytosis experiments were carried out with the J774.16 macrophage-like cellline, derived from a reticulum cell sarcoma (37), or with the MH-S line, origi-nated from an SV40-transformed adherent cell-enriched population of alveolarmacrophages (23). The protocol for in vitro phagocytosis was that described inearlier studies (46, 47) with minor modifications. Primary cells were plated on96-well tissue culture plates (Switzerland) at a final density of 1.6 � 105 cells/well,and macrophage-like cells were plated at a density of 0.8 � 105 to 1 � 105

macrophages in order to obtain the same density after overnight incubation. Thecells were stimulated with 50 U/ml recombinant murine IFN-� (PeproTech,Rock Hill, NJ) and incubated at 37°C overnight. Phagocytosis was measured inthe presence or absence of purified MAbs (0 to 100 �g/ml). P. brasiliensis cellswere added at a ratio of 5:1 macrophages to fungal cells and incubated at 37°Cfor 6, 12, and 24 h. The cells were then washed several times with sterile PBS,fixed with cold absolute methanol, and stained with a 1/20 solution of Giemsa(Sigma, St. Louis, MO). Phagocytosed yeasts were counted by light microscopyat �400 magnification. The phagocytic index (PI) is defined by the equation PI �P � F, where P is the percentage of macrophages with internalized yeast and Fis the average number of yeast cells per macrophage. Phagocytosis was confirmedby transmission electron microscopy that showed the internalization of yeast cells

in macrophages (data not shown). Experiments were carried out in triplicate, andfive to eight different fields were counted.

Intratracheal infection of BALB/c mice. BALB/c mice were inoculated i.t. with3 � 105 yeast cells of virulent P. brasiliensis Pb18, grown on Sabouraud agar andsuspended in sterile saline (0.85% NaCl), per animal. Briefly, the mice wereanesthetized i.p. with 200 �l of a solution containing 80 mg/kg ketamine and 10mg/kg of xylazine (both from Uniao Quımica Farmaceutica, Brazil). After ap-proximately 10 min, their necks were hyperextended, and the tracheas wereexposed at the level of the thyroid and injected with 3 � 105 yeast cells in PBSusing a 26-gauge needle. The incisions were sutured with 5-0 silk.

Fungal burden in organs of i.t. infected mice. For the fungal burden analysis,two protocols were utilized: (i) 1 mg of MAbs was administrated i.p. 24 h beforethe infections, and the mice were sacrificed 15 and 30 days after i.t. infection, and(ii) 1 mg of MAbs was administered i.p. 30 days after the infections, and the micewere sacrificed 45 and 60 days after i.t. infection. The fungal burden was mea-sured by CFU. Sections of the lungs, livers, and spleens were removed, weighed,homogenized, and then washed three times with PBS. The corresponding pelletswere resuspended and homogenized, each in 1 ml of PBS. A 100-�l sample ofthis suspension was plated on solid brain heart infusion medium supplementedwith 4% fetal calf serum (Gibco, NY), 5% spent P. brasiliensis (strain 192)culture supernatant, 10 IU/ml streptomycin/penicillin (Cultilab, Brazil), and 500�g/ml cycloheximide (Sigma, St. Louis, MO). The petri dishes were incubated at37°C for at least 10 days, and colonies were counted (1 colony � 1 CFU).

Histopathology. The lungs of i.t. infected mice were excised, fixed in 10%buffered formalin, and embedded in paraffin for sectioning. The sections werestained with hematoxylin-eosin or silver nitrate and examined microscopically at�25 magnification (Optiphot-2; Nikon, Tokyo, Japan).

Fungal burden in organs of i.v.-infected mice. MAbs 3E and 32H and irrele-vant MAb were administered (1 mg) i.p. 24 h before i.v. infection with 3 � 105

yeast cells of virulent P. brasiliensis Pb18. A maintenance dose of 100 �g of eachMAb was given every week for a month. The mice were sacrificed 30 days afterinfection and the fungal burden measured as described for the i.t. infection.

Cytokine analysis. Sections of the lungs (alternating right and left lungs) werehomogenized in 2 ml of PBS in the presence of protease inhibitors (CompleteMini; Boehringer Mannheim, Indianapolis, IN). The homogenates were centri-fuged, and the supernatants frozen at �80°C until tested. The supernatants wereassayed for IL-2, IL-4, IL-10, IL-12, and IFN-� using ELISA kits (BD Phar-Mingen, San Diego, CA). The detection limits of such assays were as follows: 3.1pg/ml for IL-2, 7.8 pg/ml for IL-4, 31.25 pg/ml for IL-10 and IFN-�, and 62.5pg/ml for IL-12p40, as previously determined by the manufacturer.

Production of nitric oxide. The levels of nitric oxide metabolite (nitrite) weredetermined by Griess reaction (33) in the culture supernatant of macrophageschallenged with opsonized yeasts. All determinations were performed in tripli-cate.

Peptide synthesis and purification for screening. Peptide synthesis and puri-fication were carried out at the Department of Biophysics, Universidade Federalde Sao Paulo. The amino acid sequence of P. brasiliensis gp43 glycoprotein(GenBank accession number AY005437) was used to synthesize the peptides bysolid-phase technology using the 9-fluorenylmethoxy carbonyl strategy on anautomated benchtop simultaneous multiple solid-phase peptide synthesizerPSSM8 (Shimadzu, Tokyo, Japan) with 9-fluorenylmethoxy carbonyl-protectedamino acid residues and TentaGel Rink resin (Novabiochem, San Diego, CA).Therefore, all peptides were obtained with the C-terminal carboxyl group in anamide form. All peptides were deprotected and cleaved from the resins bytreatment with K reagent composed of 80% trifluoroacetic acid, 2.5% triisopro-pylsilane, 2.5% ethanedithiol, 5.0% anisole, 5.0% water, and 5.0% phenol. Theresulting peptides were analyzed by reverse-phase high-performance liquid chro-matography (Shimadzu, Tokyo, Japan) on a C18 column eluted at 1 ml/min usinga 5% to 95% gradient of 90% acetonitrile in 0.1% trifluoroacetic acid over 30min. The peptide quality was assessed by a matrix-assisted laser desorptionionization–time of flight instrument (Micromass, Manchester, United Kingdom)using -cyano-4-hydroxy cinnamic acid as the matrix.

Peptide containing the reactive epitope of the protective MAb 3H. The peptideNHVRIPIGYWAV was synthesized at Peptides International (Louisville, KY)with 95% purity, in both the carboxy and amidated forms.

MAb reactivity with peptides. Antibody reactivity with peptides from gp43 wasdetermined by ELISA. Microtiter plates coated with 100 ng of each peptide/wellwere incubated with 100 �l of MAb serially diluted, starting at 10 �g/ml, at 37°Cfor 1 h. The plates were washed three times and incubated with 100 �l of goatanti-mouse IgG peroxidase (Sigma, St. Louis, MO) for 1 h at 37°C. The plateswere washed, and the reaction was developed with a solution of o-phenylenedi-amine (0.5 mg/ml; Sigma, St. Louis, MO) and 0.005% H2O2 (Sigma, St. Louis,MO). The reaction was terminated with 4 N H2SO4 after 8 to 10 min of incu-

3322 BUISSA-FILHO ET AL. INFECT. IMMUN.

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bation in the dark. The optical densities were measured at 492 nm in an ELISAreader (Titertek Multiskan EIA reader).

Direct effect of MAbs on P. brasiliensis. The growth rate of Pb18 in thepresence of protective and nonprotective MAbs was compared to that in thepresence of irrelevant antibodies or medium alone. Yeast cells were grown inSabouraud medium at 37°C, and 100 �g/ml of each MAb was added at 96-hintervals. Culture samples were taken at 48-h intervals, and cell numbers werecounted with a hemocytometer.

Statistical analysis. Statistical analysis was done using GraphPad Prism5 soft-ware. The results were expressed as the means the standard deviations (SD)of the indicated numbers of animals or experiments. The nonparametric Tukey’shonestly significant difference test was used. The unpaired Student’s t test withWelch’s correction (two-tailed) was used for the comparison of the two groupswhen the data met the assumptions of the t tests. P values of �0.05 indicatedstatistical significance.

RESULTS

In vitro phagocytosis mediated by MAbs. We investigatedthe effect of MAbs to gp43 on P. brasiliensis phagocytosis invitro using both primary macrophages (lung and peritoneal)and cell lines J774.16 and MH-S. Yeast cells opsonized withMAbs 19G, 21F, 10D, 17D, and 3E were at least twofold moreinternalized by J774.16 cells than by nonopsonized or 32H-opsonized yeast cells (Fig. 1). The same result was obtainedwith primary macrophages. The MH-S cell line and the lungprimary macrophages showed similar results. Phagocytosis wasobserved regardless of whether or not the macrophages wereactivated with IFN-�, but the indices were significantly higherwith cytokine-stimulated cells (data not shown).

Yeast cells opsonized with MAbs to gp43 promote macro-phage fungicidal activity in vitro. MAbs that increase P. bra-siliensis phagocytosis by macrophages also induce an increasein nitric oxide metabolite (nitrite) in the supernatants of thephagocytic cells (Fig. 2).

Passive immunization reduces fungal burden. To evaluatewhether the administration of MAbs could reduce lung CFUfrom mice infected with P. brasiliensis, two protocols werefollowed. In the first protocol, BALB/c mice were immunizedwith different MAbs against gp43 24 h prior to i.t. infectionwith 3 � 105 yeast cells of P. brasiliensis. After 15 days ofinfection, the number of CFU in the lungs of mice that receivedMAbs 19G, 21F, 10D, 3E, and 17D but not 32H showed a sig-nificant reduction compared with that of the controls (Fig. 3).Additionally, MAbs 19G, 10D, and 3E maintained significantCFU reductions after 30 days of infection (Fig. 3). In the secondprotocol, two MAbs were selected, 32H that did not reduce thelung CFU and 3E that reduced the CFU. BALB/c mice wereinfected as indicated above, and after 30 days, 1 mg of MAbs 32Hor 3E was injected i.p. The CFU were measured after 45 and 60days of infection, and MAb 3E but not 32H was able to reduce theCFU from the lungs of the mice at day 60 (Fig. 4).

FIG. 1. Phagocytosis of yeast cells by J774.16 cells after 12 h in thepresence of MAbs against gp43 (3E, 10D, 17D, 19G, 21F, and 32H)compared to that of controls of yeast incubated without MAb (PBS) orwith an irrelevant MAb (MAb A4). Each bar represents the average ofthree measurements, and error bars indicate SD. Experiments weredone in triplicate, and different fields were counted. �, significantdifference (P � 0.05, determined by analysis of variance and Tukey’shonestly significant difference test) compared to the control withoutMAb.

FIG. 2. Nitrite measurements in supernatants from phagocytosis assays. P. brasiliensis yeast was cultured with J774.16 cells for 12 h in thepresence of MAbs against gp43 (3E, 10D, 17D, 19G, 21F, and 32H), PBS (without MAb), or the irrelevant MAb (A4). Nitrite levels were detectedusing a Griess assay. �, significant difference (P � 0.05, determined by analysis of variance and Tukey’s honestly significant difference test) relativeto the PBS control. Error bars denote SD.

VOL. 76, 2008 IMMUNE PROTECTION BY ANTI-gp43 MONOCLONAL ANTIBODY 3323

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To evaluate the effect of MAbs in mice, an intravenousinfection model was used. Groups of animals were passivelyimmunized i.p. with 1 mg of MAb 3E, MAb 32H, or theirrelevant control MAb 24 h prior to infection with 5 � 106

yeast cells and were given 0.1 mg of the MAbs every week for1 month before the animals were sacrificed. For the CFUdeterminations, the lung, spleen, and liver tissues were homog-enized separately and plated on solid medium. The mice im-munized with MAb 3E had significantly lower lung and spleenCFU compared to the CFU of the mice receiving MAb 32H,the irrelevant MAb, or PBS (Fig. 5). Liver CFU were below thedetection threshold.

Viability of P. brasiliensis yeast cells in the presence of MAbs3E and 32H. The viability of yeast cells incubated with MAbs3E or 32H or the irrelevant MAb was evaluated during 30 days;no direct inhibitory effects by the MAbs were observed (datanot shown).

Lung histopathology of treated, i.t. infected BALB/c mice(30 days). The lungs of the untreated control animals showedintense infiltration, mainly by macrophages, lymphocytes, andepithelioid cells. Around the foci of the epithelioid granulo-mas, giant cells were also observed. Multiplying fungal cellswere seen. Using protocol 1 (for which the mice receivedMAbs 24 h before i.t. infection), we observed that the animals

FIG. 3. Lung CFU from mice infected i.t. with 3 � 105 yeast cells and treated with MAbs against gp43 (3E, 10D, 17D, 19G, 21F, and 32H) 24 hprior to infection. Mice were sacrificed after 15 (black bars) or 30 (gray bars) days of infection. Control mice were infected and received PBS(infected only) or an irrelevant MAb (A4). Each bar represents the average count of fungi in the lung, and error bars indicate SD. �, significantdifference (P � 0.05, determined by analysis of variance and Tukey’s honestly significant difference test) relative to the PBS control.

FIG. 4. Lung CFU from mice infected i.t. with 3 � 105 yeast cells and treated with MAbs against gp43 (3E or 32H) 30 days after infection. Micewere sacrificed after 45 (black bars) or 60 (gray bars) days of infection. Control mice were infected and received PBS (infected only) or an irrelevantMAb (A4). Each bar represents the average count of fungi in the lung, and error bars indicate SD. �, significant difference (P � 0.05) relative tothe PBS control.


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immunized with MAb 32H were pathologically similar to theinfected control mice. In contrast, the animals that receivedMAb 3E had fewer epithelioid granulomas with a reducednumber of yeast cells and large areas of preserved lung tissue(Fig. 6).

MAb treatment alters cytokine expression. Cytokine levelswere detected in the lung tissue of i.t. infected mice treatedwith MAb 3E and 32H in both protocols. Groups of 10 micewere used in each protocol; all experiments were repeatedtwice with similar results. As shown in Table 1, with the firstprotocol, animals immunized with MAb 3E had higher levelsof IFN-� and reduced levels of IL-4 after 15 and 30 days ofinfection compared with those of the animals immunized withMAb 32H. The other cytokines (IL-10, IL-12, and TNF-)showed less variability. With the second protocol, the levels ofIL-12 after 45 days of infection and IFN-� after 45 and 60 daysof infection were higher than those of MAb 32H (Table 2).

Identification of the epitope of MAb 3E. The reactivity ofMAb 3E was evaluated against a panel of gp43-derived pep-tides previously described (48). The analysis of the resultssuggested that the recognized epitope is within the sequenceNHVRIPIGWAV (data not shown). The MAb 3E but not 32H

recognized this peptide using ELISA (Fig. 7). This peptidesequence was found to be conserved in the internal sequencesof �-1,3-glucanases of Aspergillus fumigatus, Aspergillus oryzae,and Blumeria graminis.

DISCUSSION

The protective role of antibodies against fungal infections isin many cases controversial. Recently, several studies haveestablished that some antibodies are protective against fungi(31, 41, 52). MAbs or fragments of MAbs have been approvedfor the clinical evaluation of cryptococcosis (21) and candidi-asis (32). Murine MAb 18B7 directed against the capsularpolysaccharide of C. neoformans was used to evaluate thesafety and maximum tolerated dose of a therapeutic MAb. Thestudy used human immunodeficiency virus-infected patientswho had successfully been treated for cryptococcal meningitis.The MAb infusion had a half-life in the serum of approxi-mately 53 h and reduced the fungal circulating antigen (21). Inanother study, a human recombinant MAb to heat shock pro-tein 90 was used in the treatment of patients with invasivecandidiasis (32). A consensus has now emerged that the inabil-

FIG. 5. CFU from lungs (black bars) or spleens (gray bars) of mice infected i.v. with 3 � 105 yeast cells and treated with 1 mg of MAbs againstgp43 (3E or 32H) 24 h prior to infection and with 100 �g every week for a month as a maintenance dose. Mice were sacrificed after 30 days ofinfection. Control mice were infected and received PBS (infected only) or an irrelevant MAb (A4). Each bar represents the average count of fungiin the organ, and error bars indicate SD. �, significant difference (P � 0.05, determined by analysis of variance and Tukey’s honestly significantdifference test) relative to the PBS control.

FIG. 6. Histopathology of representative lung sections from mice i.t. infected according to protocol 1 and euthanized at 30 days after infection.(A) Lung section from an untreated infected mouse; (B) lung section from a mouse infected and treated with MAb 32H; (C) lung section froman infected mouse treated with MAb 3E. Hematoxylin-eosin staining, �10 magnification.


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ity of immune sera to mediate protection against fungi reflectsinadequate amounts of protective antibody and/or the simul-taneous presence of protective and nonprotective antibodiesrather than a fundamental inability of antibody to protectagainst fungal pathogens (11).

The first evidence of an antibody-mediating protectionagainst P. brasiliensis was described by de Mattos Grosso et al.(17). The authors showed that the passive transfer of twomurine MAbs against a glycoprotein of 70 kDa, which is rec-ognized in 96% of the sera from PCM patients, led to a sig-nificant reduction in the number of CFU in the lungs and fewergranulomas within the organs of experimentally infected mice(17). More recently, another MAb that recognizes a secreted75-kDa protein with phosphatase activity inhibited P. brasilien-sis growth in vitro and reduced lung CFU in vivo (52).

Although the MAbs to gp43 have not directly affected thegrowth of P. brasiliensis, the protective action of the MAbs wasdirectly associated with their capacity to enhance phagocytosis.Since macrophages are effector cells capable of exerting yeastfungicidal effects, we investigated NO production in macro-phages that had phagocytosed MAb-opsonized yeast cells. TheNO results showed an increase for all “protective” MAbs, but

the response was variable, with MAb 3E demonstrating themost impressive effect. Previous data from our group and oth-ers clearly demonstrated that activated macrophages kill thefungus in vitro by the production of nitric oxide and hydrogenperoxide (7, 8).

Using a panel of six MAbs against gp43, including IgG2a(10D, 17D, 19G, and 32H) and IgG2b (3E and 21F), we iden-tified the protective and nonprotective MAbs by the passiveadministration of the antibodies into mice infected i.t. or i.v.with P. brasiliensis. The presence of protective and nonprotec-tive antibodies against the same antigen is a conceptually rel-evant finding that has previously been described for cryptococ-cosis (27). The histopathology of lung sections from i.t.infected animals treated with the protective MAb showed areduction in the number of yeast cells and epithelioid granu-lomas in comparison with the untreated control mice.

The role of IFN-� in mediating activated macrophages in asystemic fungal infection has been reported. Murine peritonealmacrophages activated by IFN-� show enhanced fungicidalactivity with both yeast and conidial forms of P. brasiliensis(7–9). The production of IL-10 has been associated with moresevere disease in susceptible mice (19). Alveolar macrophages

TABLE 1. Protocol 1 cytokine levels in the lungs of mice in response to the passive transfer of MAbs 24 h before i.t.infection with P. brasiliensis

Cytokine Days ofinfection

Cytokine level (pg/g of tissue)a

Sham Infected only Irrelevant MAb A4 Treated withMAb 3E

Treated withMAb 32H

IL-4 15 248.2 63.1 248.7 43.6 327.1 56 102.8 52b 107.7 49.1b

30 322.5 70.7 380.4 81.9 422.4 87.8 120.6 67b 559.5 76.9b

IL-10 15 4,204 269.4 6,786.9 1,250.1 8,062.5 926.4 11,027.8 1,321.8b 12,852 1,560b

30 3,920 330 9,033.3 978.4 9,357.1 1,077.8 15,804 2,135b 24,155 1,398b

IL-12 15 4,682.1 303 8,648.6 218.2 8,543.7 743 12,829.9 1,422.5b 8,724.3 1,403.430 4,880 335.8 7,598 838 7,050 765 6,535.3 834.2 10,128.4 988b

IFN-� 15 101 13 300 35.1 240 38.3 1,100.8 115.6b 150.4 87.830 108 10.1 260.9 8.3 301.3 50.8 780 99.3b 155.7 93.4

TNF- 15 2,142.8 505 12,616.7 918.7 11,725 1,426.6 6,944.4 1,387.5b 4,217.9 657.5b

30 2,680 622.2 25,794.8 2,492.2 22,857.1 2,412 14,456.5 2,224.4b 11,662.2 2,380.1b

a Values are means standard deviations of measurements from 10 animals per group. The experiment was repeated twice with reproducible results.b Statistically significant (P � 0.05, determined by analysis of variance and Tukey’s honestly significant difference test) relative to the value for the untreated, infected

mice.

TABLE 2. Protocol 2 cytokine levels in the lungs of mice in response to the passive transfer of MAbs 30 days after i.t.infection with P. brasiliensis

Cytokine Days ofinfection

Cytokine level (pg/g of tissue)a

Sham Infected only Irrelevant MAb A4 Treated withMAb 3E

Treated withMAb 32H

IL-4 45 208.2 73.1 589.2 81.9 342.3 36.2 2,151.6 352b 1,370.6 134b

60 254.3 85.7 862.8 185.5 764.8 107.8 1,633.2 188b 1,832.9 287.6b

IL-10 45 1,685.7 252.5 2,541.9 172.2 1,666.7 250 3,245.4 318.1b 1,790.6 240.6b

60 2,360 494.7 2,760.2 612.3 3,238.1 798.4 3,583.3 279b 2,500 839.9IL-12 45 3,257.6 370.4 4,912.5 817.4 8,362.5 1,969.6 21,337.9 3,475.9b 12,470.1 1,815.5b

60 3,243.7 1,885.6 6,987.4 891.5 9,509.5 3,249.4 14,699.3 2,981.1b 11,630.6 2,350.8b

IFN-� 45 99.41 33.1 280.9 41.2 106.6 29.2 1,328.6 207.2b 162.5 42.1b

60 90.2 25.4 382.4 39.2 133.3 33 1,229.8 192.3b 416.4 59.3TNF- 45 1,980.5 359 22,870.5 1,057.6 22,100 3,225.5 16,985.4 1,698.2b 18,798.5 2,785.2b

60 2,010.5 438.8 30,021 3,777.8 26,789.2 1,469.3 14,892.4 2,001b 21,364.7 3,911.2b

a Values are means standard deviations of measurements from 10 animals per group. The experiment was repeated twice with reproducible results.b Statistically significant (P � 0.05, determined by analysis of variance and Tukey’s honestly significant difference test) relative to the value for the untreated, infected

mice.


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from i.t. infected IL-4-deficient mice more efficiently controlfungal growth than wild-type macrophages (34), and also IL-4-deficient mice have increased levels of pulmonary IFN-�(34). The passive administration of irrelevant MAb, 3E, or 32Hin i.t. infected BALB/c mice showed a mixed Th1/Th2 patternof activation. Nevertheless, the cytokine changes associatedwith protective MAb administration were more Th1-like, giventhe increased levels of IFN-� and IL-12 in the lungs of i.t.infected mice. A discreet but statistically significant increase ofIL-10 and a reduction of IL-4 and TNF- were noted com-pared with the levels of IL-10, IL-4, and TNF- in the controlinfected mice. The passive transfer of MAb 32H induced lowerlevels of lung IFN-� and IL-12 than MAb 3E treatment. Infungal disease, the effects of antibody treatment on cytokineproduction are poorly understood and may depend on severalfeatures, including the fungus, target organ, timing of fungaldissemination, and antibody quantity, isotype, and specificity(18, 39, 49). Antibody-mediated protection has been associatedwith lower levels of IFN-� in A/J mice infected with C. neo-formans (18), but both Th1- and Th2-related cytokines arenecessary for antibody protection in cryptococcosis (3). StrongTh1 responses can result in reduced survival and tissue damage(22, 39). In our studies, we found that MAb treatment did nothave the polarity of the Th1/Th2 response and that IFN-�appears to be beneficial.

Attempts at determining the epitope of protective MAb 3Eled to the sequence NHVRIPIGYWAV shared with the A.fumigatus, A. oryzae, and B. graminis internal sequences of�-1,3-glucanases. The opsonizing effect of MAb 3E could theninvolve the recognition of this peptide sequence in the gp43accumulated on the cell wall of P. brasiliensis. In fact, gp43 isstored inside a vacuole, migrates to the plasma membrane, andspreads into the P. brasiliensis cell wall. The secretion of thisantigen occurs at discrete sites along the cell surface (45).

Historically, protection against PCM has been attributed toa vigorous cellular immune response, whereas specific highlevels of antibodies have been associated with disease severity,as observed in patients with acute and subacute forms. Here we

show that there are protective and nonprotective anti-gp43antibodies and that the former play an important role in dis-ease control. Antibodies against gp43 are found in almost100% of patients with PCM (5). Probably, there is a low con-centration of protective antibodies in the serum samples ofthese patients, and they are not sufficient to control the dis-ease. To overcome this, passive immunization with MAbs togp43 was used and shown to be protective in mouse models ofPCM. The present work also raises the potential therapeuticuse of the peptide carrying the B epitope of MAb 3E in activeimmunization adjuvant to chemotherapy and/or in associationwith a peptide sequence containing the T-cell epitope of gp43,known as P10, that elicits a strong cell-mediated immunity andis protective against experimental infection.

ACKNOWLEDGMENTS

The present work was supported by FAPESP grant 05/02776-0. R.P.,L.R.T., and C.P.T. are research fellows of CNPq. J.D.N. is supportedin part by NIH AI056070-01A2.

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50. Travassos, L. R., C. P. Taborda, L. K. Iwai, E. Cunha Neto, and R. Puccia.2004. The gp43 from Paracoccidioides brasiliensis: a major diagnostic antigenand vaccine candidate, p. 279–296. In J. E. Domer and G. S. Kobayashi (ed.),The mycota, vol. 12. Human fungal pathogens. Springer-Verlag, Berlin,Germany.

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BRAGA, C. J. M.; RITTNER, G. M. G.; MUÑOZ, J. E.; TEIXEIRA, A. F.; MASSIS, L. M.; SBROGIO-ALMEIDA, M. E.; TABORDA, C. P.; TRAVASSOS, L. R.; FERREIRA, L. C. S. Paracoccidioides brasiliensis Vaccine Formulations Based on the gp43-Derived P10 Sequence and the Salmonella enterica FliC Flagellin. Infection and Immunity (Print), v. 77, p. 1700-1707, 2009.

INFECTION AND IMMUNITY, Apr. 2009, p. 1700–1707 Vol. 77, No. 40019-9567/09/$08.00�0 doi:10.1128/IAI.01470-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Paracoccidioides brasiliensis Vaccine Formulations Based on thegp43-Derived P10 Sequence and the Salmonella enterica

FliC Flagellin�

Catarina J. M. Braga,1 Glauce M. G. Rittner,1 Julian E. Munoz Henao,1 Aline F. Teixeira,1Liliana M. Massis,1 Maria E. Sbrogio-Almeida,2 Carlos P. Taborda,1

Luiz R. Travassos,3 and Luís C. S. Ferreira1*Departamento de Microbiologia, Universidade de Sao Paulo, Sao Paulo, Brazil1; Divisao de Desenvolvimento Tecnologico e Producao,

Instituto Butantan, Sao Paulo, Brazil2; and Departamento de Microbiologia, Imunologia e Parasitologia,Universidade Federal de Sao Paulo, Sao Paulo, Brazil3

Received 2 December 2008/Returned for modification 7 January 2009/Accepted 31 January 2009

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Para-coccidioides brasiliensis. Anti-PCM vaccine formulations based on the secreted fungal cell wall protein (gp43)or the derived P10 sequence containing a CD4� T-cell-specific epitope have shown promising results. In thepresent study, we evaluated new anti-PCM vaccine formulations based on the intranasal administration of P.brasiliensis gp43 or the P10 peptide in combination with the Salmonella enterica FliC flagellin, an innateimmunity agonist binding specifically to the Toll-like receptor 5, in a murine model. BALB/c mice immunizedwith gp43 developed high-specific-serum immunoglobulin G1 responses and enhanced interleukin-4 (IL-4) andIL-10 levels. On the other hand, mice immunized with recombinant purified flagellins genetically fused withP10 at the central hypervariable domain, either flanked or not by two lysine residues, or the synthetic P10peptide admixed with purified FliC elicited a prevailing Th1-type immune response based on lung cell-secretedtype 1 cytokines. Mice immunized with gp43 and FliC and intratracheally challenged with P. brasiliensis yeastcells had increased fungal proliferation and lung tissue damage. In contrast, mice immunized with the chimericflagellins and particularly those immunized with P10 admixed with FliC reduced P. brasiliensis growth and lungdamage. Altogether, these results indicate that S. enterica FliC flagellin modulates the immune response to P.brasiliensis P10 antigen and represents a promising alternative for the generation of anti-PCM vaccines.

Paracoccidioidomycosis (PCM) is a granulomatous diseasecaused by the thermodimorphic fungus Paracoccidioides bra-siliensis, which is prevalent in Brazil and other Latin Americancountries (5, 6, 33). Some individuals develop one of the twomain clinical forms of PCM. The acute form is characterized byimpaired cellular immunity, negative delayed-type hypersensi-tivity reactions, increased systemic proliferation of the fungus,and high mortality rate. The chronic form, ranging from mildto severe chronic disease, shows exacerbated host cellular im-mune responses and formation of granulomas containing fun-gal cells and may evolve to develop extensive sequelae, includ-ing fibrotic lesions and impairment of lung function (33, 39).

Vaccines against PCM are still not available for human use,but promising formulations have been experimentally testedduring the last few years. Irradiated P. brasiliensis or cellularantigens fractionated by anion-exchange chromatography con-ferred partial protection against fungal proliferation in themurine model (11). The extracellular gp43 glycoprotein, themajor diagnostic antigen of P. brasiliensis, is the most inten-sively studied component aimed at a vaccine for PCM control.Previous reports have shown that mice immunized with thepurified protein, DNA, or anti-idiotypic monoclonal antibody

were partially protected against challenges by P. brasiliensis(28, 36, 37, 40). A 15-amino-acid peptide (QTLIAIHTLAIRYAN), designated P10, contains the gp43 immunodominantCD4� T-cell-specific epitope presented by major histocompat-ibility complex class II molecules from three different mousehaplotypes (37) and most human HLA-DR alleles (17, 18).Indeed, parenteral immunization with P10 in complete Freundadjuvant (CFA), or in the form of a truncated multiple-antigenpeptide (MAP) complex, induced protective Th1 cellular im-mune responses in mice against intratracheal (i.t.) challengewith a virulent P. brasiliensis isolate (37, 38, 41).

The rational use of vaccines has been significantly improvedafter elucidation of innate immune mechanisms in mammaliancells. The recognition of distinct pathogen-associated molecu-lar patterns by members of the Toll-like receptor (TLR) familyinitiates a signaling cascade mediated by adaptor proteins,including MyD88 and interleukin-1 (IL-1) receptor-associatedkinase, that culminates in the production of proinflammatorycytokines, such as tumor necrosis factor alpha and IL-12, andincreased expression of cell surface molecules involved inepitope presentation by antigen-presenting cells (APC) (1, 19).Proper APC activation by TLR agonists represents a key stepfor an effective adaptive immune response induced by patho-gens or vaccines and explains, at least in part, the markedadjuvant effects of several bacterial molecules, including lipo-polysaccharides, lipoproteins, peptidoglycan fragments, andflagellins (2).

Flagellin, the structural subunit of bacterial flagellum, is a

* Corresponding author. Mailing address: Department of Microbi-ology-ICB, University of Sao Paulo, Av. Prof. Lineu Prestes, 1374 SaoPaulo, SP 05008-000, Brazil. Phone: 55-11-30917338. Fax: 55-11-3091-7354. E-mail: [email protected].

� Published ahead of print on 9 February 2009.

1700

highly conserved protein that induces TLR5-dependent in-flammatory responses and exerts strong adjuvant effects onboth antibody and cellular immune responses (12, 13, 30).Flagellin has been successfully used as a vaccine adjuvant togenerate antigen-specific antibodies and T cells either whenadministered to mice as the native purified protein (4, 14, 20,27) or as a hybrid protein genetically fused to the target anti-gen (10, 15, 16). Additionally, in contrast to other vaccineadjuvants, such as CFA, flagellin may exert strong adjuvant effectsfollowing administration through mucosal routes (14, 27).

In the present study, we have evaluated the adjuvant effectsand protective efficacy of intranasal (i.n.) anti-PCM vaccineformulations based on Salmonella enterica serovar Dublin FliCflagellin and purified recombinant P. brasiliensis gp43 or thesynthetic P10 peptide. In addition, recombinant chimericflagellins genetically fused to P10 were also tested as potentialanti-PCM vaccine antigens. The results demonstrate that S.enterica FliC flagellin modulates the murine immune systemfavoring either the generation of antibodies (gp43 plus FliC) oractivation of cellular immune responses. In accordance withthe administered vaccine formulation, mice challenged with P.brasiliensis were differentially protected against exacerbatedfungal proliferation. The present results indicate that Salmo-nella FliC has an important role in the generation of mucus-delivered anti-P. brasiliensis peptide-based vaccine formula-tions. Furthermore, flagellin-based adjuvants may contributeto the understanding of immune mechanisms involved in PCMdevelopment.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. The bacterial strains andplasmids used in this study are described in Table 1. The strain Salmonellaenterica pv. Dublin SL5928 is a nonflagellated aroA attenuated vaccine strain(34). Salmonella pv. Dublin SL5930 is a SL5928 derivative transformed with theplasmid pLS408 encoding the gene fliCd, originally derived from Salmonella

enterica serovar Munchen, with a 48-bp deletion generated after removal of aEcoRV-EcoRV fragment from the central hypervariable domain leading to asingle EcoRV site used for in-frame insertion of nucleotide sequences encodingheterologous epitopes. All S. Dublin and Escherichia coli strains were routinelycultivated at 37°C with shaking (200 rpm) in Luria-Bertani (LB) broth or on LBagar plates supplemented with ampicillin (100 �g/ml).

Fungal strain. The virulent P. brasiliensis Pb18 strain was maintained byweekly passages on solid Sabouraud medium at 36°C. Before experimental in-fection, cultures were grown in modified McVeigh-Morton medium at 36°C for5 to 7 days (31). The fungal cells were washed in phosphate-buffered saline (PBS;pH 7.2) and counted in a hemocytometer. The upper part of cell suspensions thatcontained isolated or single budding cells was used in the infection experimentsand for cell counting after decanting the clusters of yeast cells. The viability offungal suspensions was determined by staining with trypan blue (Sigma, St.Louis, MO) and was always higher than 90%. The virulence of the Pb18 strainwas checked in each experiment by infecting BALB/c mice i.t. and recovering theyeast cells from the infected organs.

Generation of hybrid flagellins genetically fused to P10. Chimeric Salmonellaflagellins were generated after cloning the complementary oligonucleotides intothe single EcoRV site of the pLS408-clonded FliC-encoding gene (24). Comple-mentary 45-base oligonucleotides P10fw (5�-GAA ACC CTG ATT GCG ATTCAT ACC CTG GCG ATT CGC TAT GCG AAC-3�) and P10rv (5�-CTT TGGGAC TAA CGC TAA GTA TGG GAC CGC TAA GCG ATA CGC TTG-3�),encoding P10 (QTLIAIHTLAIRYAN), were melted at 65°C, annealed by slowlycooling at room temperature, and blunt end ligated with T4 DNA ligase to theEcoRV-cleaved pLS408. The same procedure was repeated with P10KKfw andP10KKrv oligonucleotides that, in addition to the sequence encoding the P10peptide, carried on both 5� and 3� ends the sequence “AAA AAA,” encoding twoadditional lysine residues flanking the heterologous epitope (KKQTLIAIHTLAIRYANKK) genetically fused to FliC flagellin, in order to improve the proteo-lytic processing by cathepsin B and enhance epitope processing and presentationby APC (42). The resulting plasmids (pLSP10, encoding the recombinant FliCgenetically fused to P10, and pLSP10L, in which the P10 peptide is flanked bytwo lysine residues) were introduced by electroporation (using 0.2-cm electro-poration cuvettes at 600 �, 25 �F, and 1.75 kV; Gene-Pulser [Bio-Rad, Hercules,CA]) into E. coli strain DH5�, and transformants were selected on LB platescontaining ampicillin. Recombinant plasmids with the right inserts were screenedby EcoRV digestion and sequenced with the BigDye Terminator DNA sequenc-ing kit (PerkinElmer Applied Biosystems, Waltham, MA) using a 15-mer primer(5�-CCA GGT GCC TAC ACC CCG-3�) corresponding to a sequence located 50bp downstream of the EcoRV insertion site in pLS408. The recombinant plas-mids encoding fliCd genetically fused to P10 or the sequence flanked by twoadditional lysines were named pLSP10 and pLSP10L, respectively. Finally, theplasmids pLSP10 and pLSP10L were introduced into the flagellin-negative S.Dublin SL5928 strain by electroporation and the recombinant vaccine strainsnamed SLP10 and SLP10L, respectively.

Purification of Salmonella flagellins. Salmonella flagellins, comprising FliC,FliCd-P10, and FliCd-P10L, were harvested from the respective S. DublinSL5930, SLP10, and SLP10L strains cultivated in LB broth, according to apreviously described procedure (4). Briefly, flagellins were obtained after cen-trifugation of cells, suspended in PBS (pH 7.4), and sheared in a bench mixer atmaximal speed (a 1-min treatment repeated three times), followed by anothercentrifugation step to remove the bacterial cells. Broken flagellar fragments wereprecipitated with acetone, suspended in PBS, and finally, submitted to heattreatment (65°C for 30 min) to dissociate the flagellin monomers. The proteinconcentration was determined using the bicinchoninic acid assay (Pierce, Rock-ford, IL), and the purity of the protein preparations was monitored by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Removal ofcontaminating lipopolysaccharide was accomplished with Detoxi-Gel columns(Pierce, Rockford, IL) according to the manufacturer’s instructions. Endotoxinlevels, determined with the chromogenic Limulus amebocyte lysate assay (Cam-brex Bio Science, Walkersville, MD), were always below 3.0 endotoxin units/�gof protein.

Purification of gp43 antigen. P. brasiliensis Pb18 was cultivated in yeast Sab-ouraud liquid medium for 7 days at 36°C with shaking. The culture was inacti-vated by adding 0.2 g of thimerosal (Merthiolate)/liter filtered through filterpaper, concentrated in a vacuum at 40°C, and dialyzed against distilled water.Purification of gp43 was obtained by affinity chromatography on Affi-Gel (Bio-Rad, Hercules, CA) bound to anti-gp43 monoclonal antibody as previouslydescribed (29). Elution was carried out with 50 mM citrate buffer, pH 2.8. Theeluate was concentrated in Amicon 10K cells, and the antigen preparation wasmonitored by SDS-PAGE revealed with silver staining. The protein content wasdetermined by the Bradford method (3).

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Relevant characteristic(s) Source orreference

StrainsSalmonella Dublin

SL5928 aroA his fliC::Tn10 24SL5930 SL5928 with pLS408; mobile 24SLP10 SL5928 with pLSP10 This studySLP10L SL5928 with pLSP10L This study

P. brasiliensisPb18 Highly virulent human isolate of

P. brasiliensis35

PlasmidspLS408 pUC19 derivative carrying the

fliCd gene from S. Muenchenwith 48-bp deletion in thehypervariable region of theFliC gene bla

24

pLSP10 pLS408 derivative with a 45-bpinsert encoding the P10 epitope

This study

pLSP10L pLS408 derivative with a 57-bpinsert encoding the P10 epitopewith two flanking lysines

This study

VOL. 77, 2009 FLAGELLIN AS ADJUVANT FOR ANTI-P. BRASILIENSIS VACCINES 1701

SDS-PAGE. SDS-PAGE was performed following standard procedures usinga Mini-Protean II vertical electrophoresis unit (Bio-Rad, Hercules, CA). Pro-teins sorted in 12% polyacrylamide gels were stained with Coomassie blue.

Immunization regimens. Isogenic pathogen-free 8- to 12-week-old maleBALB/c (H-2d) mice were supplied by the Isogenic Mouse Breeding Facility ofthe Department of Parasitology, Institute of Biomedical Sciences (ICB), Uni-versity of Sao Paulo (USP). Animal handling was carried out in accordance withthe Brazilian code for use of laboratory animals and was approved by the EthicsCommittee of ICB, USP. Groups of 8 to 10 animals were immunized via the i.n.route with 15 �g of each fusion protein (FliCd-P10 or FliCd-P10L) or with amixture of 20 �g P10 peptide or 25 �g gp43 protein with 5 �g FliC flagellin.Control groups were immunized with sterile PBS, 25 �g of gp43, 5 �g FliCflagellin, or 20 �g of P10 peptide in Freund adjuvant 1:1 (vol/vol). The formu-lations were instilled into the nostrils (5 �l/nostril) with a micropipette on days0, 21, and 28. The control group immunized with P10 in Freund adjuvantreceived 1 dose (20 �g of the peptide plus CFA in a total volume of 50 �l) viathe subcutaneous route (rear footpads) and 3 doses (20 �g of the peptide inincomplete Freund adjuvant; a total volume of 200 �l) administered intraperi-toneally at 1-week intervals. Immune responses were evaluated 7 days afterimmunization and 60 days after fungal challenge.

Challenge with P. brasiliensis yeast cells. Eighty days after the last immuniza-tion, mice were inoculated i.t. with 3 � 105 yeast cells/animal of virulent P.brasiliensis Pb18 grown on Sabouraud agar and suspended in sterile saline(0.85% NaCl). A maximum of 50 �l was inoculated per mouse. Briefly, mice wereanesthetized intraperitoneally with 200 �l of a solution containing 80 mg ofketamine/kg of body weight and 10 mg/kg of xylazine (both from Uniao QuímicaFarmaceutica, Brazil). After approximately 10 min, the necks were extended andthe tracheas exposed at the level of the thyroid. For the i.t. inoculation, a26-gauge needle was used and the incisions were sutured, right afterwards, witha 5 to 0 silk thread.

Fungal burden in organs of infected mice. Mice were sacrificed 60 days afteri.t. infection, and the fungal burden was measured by CFU. Sections of the lungs,livers, and spleens were removed, weighed, and homogenized using a tissuegrinder in 10 ml of sterile PBS. The corresponding pellets were resuspended andhomogenized each in 1 ml of PBS. A 100-�l sample of this suspension was platedon solid brain heart infusion medium supplemented with 4% fetal calf serum(Gibco, NY), 5% spent P. brasiliensis (strain 192) culture supernatant, strepto-mycin/penicillin (10 IU/ml) (Cultilab, Brazil), and cycloheximide (500 mg/ml)(Sigma, St. Louis, MO). Petri dishes were incubated at 36°C for at least 20 days,and colonies were counted (1 colony � 1 CFU) (35).

Lung histopathology. Following immunization with different vaccine formula-tions, BALB/c mice were i.t. infected and sacrificed after 2 months. The lungswere excised, fixed in 10% buffered formalin, and embedded in paraffin forsectioning. The sections were stained with hematoxylin-eosin and examinedmicroscopically (Optiphot-2; Nikon, Tokyo, Japan).

Cytokine determination by enzyme-linked immunosorbent assay. Cytokineanalysis was performed 7 days after the immunizations and 2 months afterinfection of the animals. Lung sections (right and left alternating) of mice werehomogenized in 2 ml of PBS in the presence of protease inhibitors (BoehringerMannheim, Indianapolis, IN). The homogenates were centrifuged, and the su-pernatants were frozen at �80°C until tested. The supernatants were assayed forIL-4, IL-10, IL-12, and gamma interferon (IFN-) using enzyme-linked immu-nosorbent assay kits (BD PharMingen, San Diego, CA). The detection limits ofsuch assays were as follows: 7.8 pg/ml for IL-4, 31.25 pg/ml for IL-10 and IFN-,and 62.5 pg/ml for IL-12p40, as indicated by the manufacturer.

Statistical analysis. Data were analyzed by one-way analysis of variance andStudent’s t test followed by Tukey’s honestly significant difference test andDunnett’s multiple comparison tests to compare the differences between themean values of the immunization groups studied.

RESULTS

Mice immunized with purified gp43 admixed with Salmo-nella FliC flagellin develop specific antibody responses. MaleBALB/c mice received three i.n. doses of purified gp43 aloneor admixed with purified FliC, and the serum gp43-specificimmunoglobulin G (IgG) responses were measured 7 daysafter the last immunization and 60 days after the i.t. challengewith P. brasiliensis strain Pb18. Enhanced gp43-specific IgG1antibodies were detected in noninfected mice immunized withthe FliC-containing vaccine formulation (IgG1 titer, 3,804 28.83) but not in animals immunized with purified gp43 (IgG1titer, 207 75.12) (Fig. 1A). A further 6.2-fold increase in theanti-gp43 serum IgG1 was detected in vaccinated mice follow-ing challenge with Pb18 (average IgG1 titer, 23,924 21.00) inmice immunized with the FliC-containing vaccine formulation

FIG. 1. Characterization of the specific serum IgG subclass response elicited in mice i.n. immunized with gp43 or gp43 admixed with FliC.(A) Anti-gp43-specific IgG subclass detected 1 week after the last immunization dose. (B) Anti-gp43-specific IgG subclass detected in vaccinatedmice 2 months after i.t. challenge with the Pb18 strain. gp43, mice immunized with 3 doses of purified gp43; gp43 � FliCd, mice immunized withpurified gp43 admixed with FliC. For the other groups (FliCd, FliCd-P10, FliCd-P10L, and P10 � FliCd), a gp43-specific antibody response wasnot detected. Values are means of endpoint titers plus standard deviations for serum pools (n � 8) prepared from each mouse group. Data arerepresentative of two independent experiments with similar results. The IgG1/IgG2a ratio of each immunization group is indicated at the top ofthe figure. Asterisks indicate a statistically significant difference observed between mice immunized with gp43 and mice immunized with gp43admixed with FliC (P � 0.01).

1702 BRAGA ET AL. INFECT. IMMUN.

(Fig. 1B). Determination of the specific IgG subclasses elicitedin mice immunized with gp43/FliC vaccine formulation re-vealed a biased type 2 response with IgG1/IgGa ratios of 7.5and 14.3 before and after the fungus challenge, respectively(Fig. 1). These results indicate that Salmonella FliC flagellinacted as a strong antibody-inducing adjuvant when admixedwith purified P. brasiliensis gp43 following mucosal administra-tion in BALB/c mice.

Generation of vaccine formulations based on P10 geneti-cally fused or admixed with Salmonella FliC. Additional anti-P.brasiliensis vaccine formulations were tested and employed theP10 peptide in combination with FliC. The first approach re-lied on the generation of hybrid flagellins in which the P10minigene was wedged in frame at the central hypervariabledomain of FliC, a permissive insertion site that did not affectboth the inflammatory and adjuvant properties of Salmonellaflagellin (Fig. 2A). The resulting recombinant S. Dublin SLP10(encoding FliCd-P10) and SLP10L (encoding FliCd-P10L)strains were nonmotile in semisolid agar plates but expressedabundant extracellular flagella composed by hybrid flagellinswith slightly altered molecular weights, as demonstrated bySDS-PAGE of purified flagella (Fig. 2B). After purification,the flagellin preparations were �95% pure and endotoxin lev-els were below 3.0 endotoxin units/�g of protein. Another

P10-based vaccine formulation involved admixing of purifiednative FliC (5 �g) with the synthetic P10 peptide (20 �g).

Cytokine expression patterns in mice immunized with dif-ferent vaccine formulations containing Salmonella FliC flagel-lin. The cytokine (IL-4, IL-10, IL-12, and IFN-) expressionpattern in the lung tissue of mice immunized with the differentvaccine formulations was determined 7 days after the last im-munization dose and 60 days following the i.t. challenge with P.brasiliensis Pb18 yeast forms. As shown in Table 2, the concen-trations of IL-4 and IL-10 were low in lung extracts from allimmunized groups except in animals immunized with purifiedgp43 admixed with FliC, in which IL-4 and mainly IL-10 ap-peared in higher concentrations than in nonimmunized mice ormice vaccinated with FliC only. This picture changed consid-erably when the same cytokines were measured in vaccinatedmice 2 months after the challenge with the Pb18 strain. TheIL-4 and IL-10 concentrations in mice immunized with gp43were approximately 30% lower than the values detected innonimmunized animals, but the addition of FliC enhancedproduction of cytokines to levels similar to those found in thenonimmunized control group. Mice immunized with the re-combinant hybrid P10/flagellins had IL-4 and IL-10 levels sim-ilar to animals immunized only with gp43 or FliC. On the otherhand, mice immunized with FliC admixed with the synthetic

FIG. 2. Generation of recombinant hybrid flagellins genetically fused to the CD4� T-cell-specific gp43-derived P10 epitope. (A) Schematicrepresentation of the recombinant flagellins after P10 in-frame insertion at FliC central hypervariable domain. The two recombinant flagellinscarried the P10 peptide (FliCd-P10) or the P10 peptide with lysine residues on each side of the fusion site (FliCd-P10L). (B) Coomassieblue-stained 12% polyacrylamide gel loaded with flagellins extracted from different Salmonella strains. Lane 1, molecular mass markers (Fer-mentas); lane 2, FliCd flagellin extracted from S. Dublin strain SL5930 (with no insert); lane 3, FliCd-P10 flagellin extracted from S. Dublin strainSLP10; lane 4, FliCd-P10L flagellin extracted from S. Dublin strain SLP10L; lane 5, purified gp43. Each well was loaded with approximately 2 �gof purified flagellins or gp43.

TABLE 2. Cytokine levels detected in lungs of mice immunized with different vaccine formulationsa

Vaccine group

Level (ng/ml) (mean SD) of cytokine:

IL-4 IL-10 IL-12 IFN-

BC AC BC AC BC AC BC AC

PBS 0.08 0.03 6.22 0.41 0.07 0.07 14.42 1.95 0.25 0.44 25.90 3.86 0.05 0.03 4.37 0.53FliCd 1.50 0.24 2.93 0.64 2.76 0.29 7.52 1.97 5.33 1.34 22.00 1.56 1.23 0.07 9.96 1.08gp43 0.20 0.01b 4.77 0.55b 0.18 0.10b 10.25 2.09 3.07 0.46b 30.07 1.44b 0.66 0.09b 12.62 0.44b

gp43 � FliCd 3.01 0.82b 6.05 1.16b 4.10 0.39c 14.40 4.90b 7.46 0.46b 36.24 2.05c 2.25 0.49b 20.61 4.13c

FliCd-P10 0.82 0.56 3.21 0.83 2.39 0.15 9.48 2.30 4.38 0.97 30.67 3.10b 1.26 0.25 4.89 1.05FliCd-P10L 0.60 0.30b 3.16 0.22 1.00 0.70b 8.95 0.93 3.65 0.83 22.76 2.26 0.44 0.06b 7.16 1.40b

P10 � FliCd 1.50 0.49 2.80 0.06 1.47 0.88b 1.46 0.66b 9.40 0.86c 36.63 2.37c 2.35 0.19b 18.10 0.25b

a Immunization groups (5 to 8 animals/group) are as described in the legend to Fig. 2. BC, before challenge; AC, after challenge.b P � 0.05; statistically significant difference relative to that of mice immunized only with the FliCd flagellin.c P � 0.01; statistically significant difference relative to that of mice immunized only with the FliCd flagellin.


P10 peptide had IL-4 and IL-10 values equal to those in non-challenged vaccinated animals, which were statistically differ-ent from the values in animals immunized only with FliC.

Measurements of IL-12 and IFN- in vaccinated miceshowed that all tested vaccines led to enhanced cytokine ex-pression in lung cells, but statistically significant differenceswith regard to nonimmunized animals were observed only inanimals immunized with gp43 and P10 admixed with FliC (Ta-ble 2). Interestingly, there was no significant enhancement inIL-12 and IFN- levels detected in mice immunized with therecombinant flagellins compared to mice immunized only withFliC, both before and after i.t. challenge with Pb18. However,mice immunized with the P10 and FliC mixture, as well asthose immunized with gp43 plus FliC, produced enhancedIL-12 and IFN- levels similar to those detected in mice im-munized with gp43 admixed with FliC. On the other hand, theIL-10 levels of P10 plus FliC-vaccinated mice remained lowerthan those detected in mice immunized with FliC, particularlyafter challenge with Pb18. Collectively, these data suggest thatthe vaccine formulation based on P10 admixed with FliC in-duced a predominant Th1 immune response compared to miceimmunized with the other tested vaccine formulations. Deter-mination of the IFN-/IL-4 (or IFN-/IL-10) and IL-12/IL-4(or IL-12/IL-10) ratios clearly demonstrated that mice immu-nized with P10 admixed with FliC developed a more pro-nounced Th1-biased immune response compared to animalsimmunized with the other vaccine formulations (Fig. 3).

Growth of P. brasiliensis in lung tissues of mice vaccinatedwith the different vaccine formulations. The protective effectsof the vaccine formulations were determined 2 months afterthe i.t. challenge with the virulent Pb18 strain. CFU countswere determined in homogenized lung tissue from vaccinatedanimals as well as from nonimmunized mice and those inocu-lated only with purified FliC (Fig. 4). In mice i.n. immunizedwith gp43 only, there was a significant reduction in the numberof fungal colonies compared to the nonimmunized (PBS) orthe FliC-immunized groups, in agreement with previously de-scribed results based on parenteral immunizations (37). Miceimmunized with gp43 admixed with FliC, however, showedenhanced fungal proliferation in the lung tissues compared tothe nonimmunized (PBS) or the FliC-immunized groups. Miceimmunized with the hybrid flagellins (FliCd-P10 or FliCd-P10L) showed a reduction in the number of viable yeast cellssimilar to that observed in mice immunized with gp43 withoutadjuvant. Furthermore, mice immunized with the vaccine for-mulation prepared with the synthetic P10 peptide admixedwith FliC had a drastically reduced number of viable fungalcells recovered from their lung tissues (�100 CFU/g lung tis-sue) 2 months after challenge with the Pb18 strain. In contrastto mice immunized with gp43 or gp43 plus FliC, no viable yeastcells were recovered from spleen and liver homogenates frommice vaccinated with P10 admixed with FliC or the hybridflagellins (data not shown).

FIG. 3. Cytokine relative ratios measured in lungs of mice after challenge with the Pb18 strain. All cytokines were measured in whole extractsof lung cells. Immunization groups and specific cytokine values used in the cytokine ratio determination were as depicted in Table 2.


Histological analysis of lung tissue damage in immunizedmice challenged with P. brasiliensis. Histopathological analysesof lung tissue samples collected from mice submitted to differ-ent vaccine formulations showed that mice immunized withgp43 admixed with FliC had extensive tissue destruction, withabundant cellular infiltration (Fig. 5B). Exudative epithelioidgranulomas containing giant cells with multiplying fungal cellswere also observed in this vaccination group to a higher extentthan in nonimmunized animals (Fig. 5A). Mice immunizedwith the hybrid recombinant FliCd-P10 and FliCd-P10L flagel-lins showed lower numbers of exudative lesions than thosedetected in animals immunized with FliC only or nonimmu-nized animals (Fig. 5C). The number of visible, detectable, andviable fungi was also lower than in the control groups. Incontrast, lung tissues of mice immunized with P10 admixedwith flagellin were virtually devoid of granulomas and viablefungal cells. The lung tissues of mice immunized with P10 plusFliC showed preserved alveolar organization with no indica-tion of phagocytic cell infiltration (Fig. 5D).

DISCUSSION

In the present study, mucus-delivered vaccine formulationsbased on the Salmonella FliC flagellin revealed contrasting, butrather interesting, results regarding induction of specific im-mune response and prophylactic protection against a virulentP. brasiliensis strain in the murine model. While i.n. adminis-tration of purified gp43 admixed with FliC induced a Th-2-biased immune response, leading to exacerbated fungal multi-plication and tissue damage, vaccine formulations based onhybrid flagellins genetically fused with the gp43-derived P10sequence, and mainly purified FliC admixed with synthetic P10peptide, resulted in a Th1 predominant immune response and

enhanced protection to the fungal challenge in vaccinatedmice. The present evidence represents the first attempt todevelop experimental anti-PCM mucosal vaccines and extendsthe knowledge about Salmonella flagellin adjuvant effects as-sociated with different antigens.

The mucus-delivered vaccines have several advantages overconventional parenteral vaccines. For example, mucosal vac-cines are easier to administer, lack iatrogenic infection risks,and more importantly, induce broader immunity, includingactivation of both systemic and local immune responses, afeature that may be particularly relevant for airborne infec-tions. Therefore, the incorporation of Salmonella flagellin, apotent adjuvant known to act both parenterally and at mucosalsites, to P10-based vaccine formulations represented an alter-native to the Freund adjuvant previously used in other anti-PCM vaccines. Indeed, it opens a renewed perspective for afuture clinical use.

The epitope-based vaccine concept was designed as a strat-egy to preserve antigen immunogenicity but avoid potentialundesirable effects, such as activation of suppressive immuneresponses or induction of self-reacting antibodies (21). The lowimmunogenicity of synthetic peptides, which represents a ma-jor drawback in the development of effective peptide-basedvaccines, stimulated parallel experimental procedures, such asthe use of potent adjuvants, synthesis of tandem repeats orMAPs, and genetic fusion with carrier proteins (25). In thepresent study, we have shown that the combination of thesynthetic P10 peptide and the Salmonella FliC flagellin elicitedstrong activation of CD4� T-cell-dependent immune re-sponses leading to the efficient control of fungal infection invaccinated mice. The best results were achieved with the pep-

FIG. 5. Representative histopathology of lung lesions caused by P.brasiliensis strain Pb18 in mice immunized with different vaccine for-mulations. Tissue samples were collected 2 months after i.t. challengewith strain Pb18. (A) Lung section from PBS-immunized mouse withgranuloma containing multiple viable fungal cells. (B) Lung sectionfrom a mouse immunized with gp43 admixed with FliC. Observe theextensive granulomatous lesions with intense cellular infiltration andlarge number of multiplying fungal cells. (C) Lung section from mouseimmunized with the hybrid FliCd-P10L. (D) Lung section from mouseimmunized with P10 admixed with FliC showing preserved alveolarstructure and absence of granulomatous lesions and fungal cells. Allsections were amplified 40-fold and stained with hematoxylin-eosin.

FIG. 4. Detection of viable fungal cells in lung tissues of vaccinatedmice after i.t. challenge with P. brasiliensis Pb18. (A) Detection ofviable fungi in mice i.n. immunized with purified gp43 or gp43 admixedwith FliC. Immunization groups are as described in the legend to Fig.2. (B) Detection of viable fungal cells in mice i.n. immunized with P10epitope genetically fused with flagellin (FliCd-P10 or FliCd-P10L) orsynthetic P10 peptide admixed with FliC flagellin. PBS, mice immu-nized only with PBS; FliCd, mice immunized only with FliC; FliCd-P10and FliCd-P10L, mice immunized with purified recombinant FliCd-P10 and FliCd-P10L, respectively; P10�FliCd, mice immunized withP10 admixed with FliCd. All mice groups were i.t. challenged with thePb18 strain and sacrificed 2 months later for determination of CFU inhomogenized lung tissue. The same experiments were repeated threetimes. Each bar represents the medium number plus standard devia-tion in organs collected from 8 to 10 animals in each group. Asterisksindicate statistically significant differences between results detected inmice immunized with gp43 and P10 and those in mice immunized onlywith FliC (*, P � 0.05; **, P � 0.01).


tide admixed with the adjuvant, thus avoiding complex andexpensive chemical synthetic procedures or generation andpurification of hybrid peptides by genetic engineering methods.The rather promiscuous binding of P10 to several major his-tocompatibility complex class II molecules, both from mice andhumans (17, 37), in combination with the strong mucosal ad-juvant effects of Salmonella flagellins resulted in enhancedvaccine efficacy, leading to more efficient control of fungusmultiplication.

The genetic fusion of ovalbumin or the influenza virus M2protein to Salmonella flagellins was required to generate anti-gen-specific B- and T-cell-dependent responses (15, 16). In ourhands, coadministration of flagellin and the P10 peptide re-sulted in a higher Th1-biased immune response than that inmice immunized with hybrid flagellin genetically fused withP10. Linking antigens to flagellin would supply in a singlemolecule the signals required for activation and maturation ofAPC, but the present results based on a mucus-delivered for-mulation as well as other parenterally delivered vaccines (4,23) clearly show that genetic fusion of flagellin to the targetantigen does not represent a requirement for proper stimula-tion of the immune system by flagellin-containing vaccineformulations.

Quantification of IFN- and IL-12 indicated that mice im-munized with P10 admixed with FliC developed a more pro-nounced Th1 immune response than mice immunized with therecombinant hybrid flagellins (FliCd-P10 and FliCd-P10L).Additionally, determination of the IFN-/IL-4 and IFN-/IL-10 ratios (as well as the IL-12/IL-4 and IL-12/IL-10 ratios)showed that mice immunized with FliC and P10 elicited apredominant Th1 immune response to other immunizationgroups, including mice immunized with gp43 and FliC andthose immunized with the recombinant hybrid P10-containingflagellins. Indeed, induction of a Th1-biased immune responsepositively correlated with asymptomatic and mild forms ofPCM in humans as well as resistance to P. brasiliensis infectionin mice (9, 22, 26, 32). Additionally, the lack of anti-gp43antibodies in animals immunized with the P10/FliC formula-tion further showed that immunization with P10 peptide avoidsother gp43 sequences involved in nonprotective anti-P. brasil-iensis immune responses. The possibility to add another gp43-derived peptide, which reacts with a protective monoclonalantibody (7), may further enhance the efficacy of the vaccineformulation by means of a proper Th1 response in combina-tion with a protective antibody response.

Innate immunity has a pivotal role on the control of P.brasiliensis replication, as well as other microbial pathogens, indifferent mammalian hosts (8). TLR ligands directly interactwith macrophages and dendritic cells, leading to inflammatoryresponses required for the direct elimination of the pathogenand generation of protective adaptive responses. In this study,we demonstrated that incorporation of Salmonella FliC, aTLR-5 ligand, may trigger anti-PCM immune responses rang-ing from complete prophylactic protection to exacerbated par-asite multiplication according to the nature of the antigentested, thus offering new tools for the understanding of theimmunological mechanisms leading to resistance or sensitivityto P. brasiliensis. In addition, the evidence that the immuno-genicity of epitope-based vaccines, when associated with Sal-monella FliC, may elicit a protective immune response in vac-

cinated mice raises new perspectives for the development ofimproved vaccine formulations and warrants further studiesaimed at the prophylactic and therapeutic control of PCM.

ACKNOWLEDGMENTS

This work was supported by a grant from Fapesp (Sao Paulo StateFoundation for Research Support) and a Brazilian federal governmentgrant for the Millennium Institute for Vaccine Development and Tech-nology (CNPq).

We acknowledge the valuable technical assistance of L. C. Silva.

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Editor: A. Casadevall


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Muñoz J. E

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Artigo 5

AMARAL, A. C.; MARQUES, A. F.; MUÑOZ, J. E; BOCCA, A. L.; SIMIONI, A. R.; TEDESCO, A. C.; MORAIS, P. C.; TRAVASSOS, L. R.; TABORDA, C. P.; FELIPE, M.S.S. Poly (lactic acid-glycolic acid) nanoparticles markedly improve immunological protection provided by peptide P10 against murine paracoccidioidomycosis. British Journal of Pharmacology, p. 1-7, 2010.

RESEARCH PAPER

Poly(lactic acid-glycolic acid) nanoparticles markedlyimprove immunological protection provided bypeptide P10 against murine paracoccidioidomycosisbph_

617 1126..11321126..1132

André C Amaral1,2, Alexandre F Marques3*, Julián E Muñoz3, Anamélia L Bocca1,Andreza R Simioni4, Antonio C Tedesco4, Paulo C Morais5, Luiz R Travassos6, Carlos P Taborda3

and Maria Sueli S Felipe1

1Biological Sciences Institute and 5Physics Institute, Universidade de Brasília, Brasília, 2Genomic Science and Biotechnology,Universidade Católica de Brasília, Brasília, 4Chemistry Institute, Universidade de São Paulo, Ribeirão Preto, 3BiomedicalSciences Institute and Laboratory of Medical Mycology-LIM53/IMTSP, Universidade de São Paulo, São Paulo, and6Microbiology, Immunology and Parasitology Department, Universidade Federal de São Paulo, São Paulo, Brazil

Background and purpose: The present study reports on the preparation and testing of a sustained delivery system for theimmunomodulatory peptide P10 aimed at reducing the in vivo degradation of the peptide and the amount required to elicita protective immune response against paracoccidioidomycosis.Experimental approach: BALB/c mice were infected with the yeast Paracoccidioides brasiliensis to mimic the chronic form ofparacoccidioidomycosis. The animals were treated daily with sulfamethoxazole/trimethoprim alone or combined with peptideP10, either emulsified in Freund’s adjuvant or entrapped in poly(lactic acid-glycolic acid) (PLGA) nanoparticles at differentconcentrations (1 mg, 5 mg, 10 mg, 20 mg or 40 mg·50 mL-1). Therapeutic efficacy was assessed as fungal burden in tissues andthe immune response by quantitative determination of cytokines.Key results: Animals given combined chemotherapy and P10 nanotherapy presented a marked reduction of fungal load in thelungs, compared with the non-treated animals. After 30 days of treatment, P10 entrapped within PLGA (1 mg·50 mL-1) wasmore effective than ‘free’ P10 emulsified in Freund’s adjuvant (20 mg·50 mL-1), as an adjuvant to chemotherapy. After treatmentfor 90 days, the higher doses of P10 entrapped within PLGA (5 or 10 mg·50 mL-1) were most effective. Treatment with P10emulsified in Freund’s adjuvant (20 mg·50 mL-1) or P10 entrapped within PLGA (1 mg·50 mL-1) were accompanied by high levelsof interferon-gamma in lung.Conclusions and implications: Combination of sulfamethoxazole/trimethoprim with the P10 peptide entrapped within PLGAdemonstrated increased therapeutic efficacy against paracoccidioidomycosis. P10 incorporation into PLGA nanoparticlesdramatically reduced the peptide amount necessary to elicit a protective effect.British Journal of Pharmacology (2010) 159, 1126–1132; doi:10.1111/j.1476-5381.2009.00617.x; published online 5February 2010

Keywords: immunomodulatory peptide; antifungal therapy; biodegradable polymers; drug delivery; nanobiotechnology

Abbreviations: CFUs, colony-forming units; DMSA, dimercaptosuccinic acid; Gp43, 43 kDa glycoprotein; IFN-g, interferon-gamma; IL-4, interleukin-4; IL-10, interleukin-10; IL-12, interleukin-12; MAP, multiple antigenic peptide; P10,peptide P10; PBS, phosphate-buffered saline; PCM, paracoccidioidomycosis; PGA, poly-glycolic acid; PLA,poly-lactic acid; PLGA, poly(lactic acid-glycolic acid)

Introduction

Extensive research has focused on the design of efficient adju-vants to vaccines for immunological protection in severaldiseases. More recently, because of the dramatic increase inthe incidence of systemic mycoses, attention has been given

Correspondence: Maria Sueli S Felipe, Universidade de Brasília, Depto de Bio-logia Celular, Laboratório de Biologia Molecular, Campus Darcy Ribeiro, AsaNorte, Brasília DF 70910-900, Brazil. E-mail: [email protected]*Present address: Department of Biological Sciences, The Border BiomedicalResearch Center, University of Texas at El Paso, El Paso, TX 79902, USA.Received 4 June 2009; revised 7 October 2009; accepted 9 October 2009

British Journal of Pharmacology (2010), 159, 1126–1132© 2010 The AuthorsJournal compilation © 2010 The British Pharmacological Society All rights reserved 0007-1188/10www.brjpharmacol.org

to the development of antifungal vaccines (Cutler et al.,2007). Peptide antigens, among other immunogens, are espe-cially promising for triggering effective immune protectiveresponses against these infections (Taborda et al., 1998;Travassos et al., 2008a). Incorporation of peptides into con-trolled release systems is an approach used to avoid degrada-tion and promote their release at predetermined rates(Johansen et al., 2000).

Biodegradable polymers are attractive delivery systems forvaccines because of their property of slowly and graduallycontrolling the release of an antigen (Commandeur et al.,2006). After in vivo administration of biocompatible poly-mers, they are broken down into molecules that take part innormal metabolic pathways and are then eliminated. Anypolymer-entrapped molecule (a drug or a peptide) is releasedupon polymer biodegradation, which depends on polymerconstitution and occurs under certain conditions, such as pHand temperature changes (Commandeur et al., 2006; Vicentand Duncan, 2006).

Polymeric systems, such as nano- and micro-particles, areappropriate as adjuvants to prepare a single-shot vaccine.Antigens encapsulated within polymers can be released for aprolonged period at a controlled rate of polymer degradation(Vicent and Duncan, 2006). Polymers have been shown to beeffective adjuvants for various antigenic proteins, includingovalbumin, cholera and tetanus toxoid, and malarial andpneumotrophic bacterial antigens (Dhiman and Khuller,1998; Jaganathan et al., 2005). Poly(lactic acid-glycolic acid)(PLGA) is a polymer whose degradation rate and antigenrelease can be predicted (Jiang et al., 2005). By releasing animmunomodulatory peptide at a predetermined rate, it cansignificantly reduce the amount and number of doses ofantigen required for protection.

Taborda et al. (1998) identified a 15-amino acid peptide thatcarries the T-cell epitope of the glycoprotein 43 kDa glycopro-tein (Gp43), the major diagnostic antigen secreted by Para-coccidioides brasiliensis (Puccia et al., 1986). This peptide,named P10, elicits an interferon-gamma (IFN-g)-dependentimmune protection against experimental paracoccidioidomy-cosis (PCM; Taborda et al., 1998; Travassos et al., 2004).Immunization with P10 improved the therapeutic efficacyagainst PCM when combined with sulfamethoxazole/trimethoprim, among other antifungals, suggesting an impor-tant contribution of P10 in improving outcome and reducingthe time of treatment against PCM (Marques et al., 2006).

Paracoccidioidomycosis is a health problem in LatinAmerica, where around 10 million individuals may beinfected by the dimorphic human pathogenic fungus P. bra-siliensis and 2% of them may develop acute or chronicforms of PCM (Brummer et al., 1993). The acute forminvolves the lymphoreticular system and may be lethal; inchronic PCM, the lung is mainly affected with a granulo-matous inflammatory response, which represents an effec-tive defence against fungal spread (Brummer et al., 1993; deCamargo and de Franco, 2000). The usual therapy for PCMis based on polyenes, azoles and sulphonamides. Amphot-ericin B is indicated in severe disseminated cases and mustbe followed by a prolonged treatment with azoles andsulfamethoxazole/trimethoprim (Brummer et al., 1993;Lortholary et al., 1999).

The present study reports on the design, preparation andin vivo testing of a sustained delivery system containingthe immunoprotective peptide P10 loaded on nanoparticlesof the biodegradable polymer PLGA. This preparation wasadministered in combination with sulfamethoxazole/trimethoprim during the treatment of a murine model ofsystemic PCM.

Methods

Preparation of PLGA particles loaded with P10 peptideThe particles were prepared using PLGA polymeric blendswith 50:50 poly-lactic acid (PLA) : poly-glycolic acid (PGA).The polymers were first dissolved in dichloromethane andthen added to an aqueous solution containing 1% polyvinylalcohol and the P10 peptide, aiming for the final preparationto contain 1, 5, 10, 20 or 40 mg·50 mL-1 of the peptide. Themixture was submitted to vigorous agitation in a blender(10 000–15 000 rpm) to obtain the water-in-oil emulsifica-tion. The organic solvent was removed from the solution bystirring at room temperature and evaporation under reducedpressure. The particles were centrifuged (4–10°C; 1100–4600¥g.) for 10- to 20-min intervals. The preparation was washedthree times in distilled water, suspended in 1.0 mL phosphate-buffered saline (PBS), stored at 4°C and used for up to 1 week.All procedures were performed in a sterile room with all themanipulations in a sterile hood.

AnimalsAll animal handling and experimental procedures performedin this study were approved by the Animal Care and UseCommittee of the Universidade de São Paulo. Male BALB/cmice (6–8 weeks old) from the Universidade de São Paulo(USP), Brazil, were used in this study. Animals were housed inpolypropylene cages under specific pathogen-free conditionsand were provided with food and water ad libitum.

Fungal inoculum for in vivo experimentsP. brasiliensis, isolate Pb18, was used to infect the animals. Thestrain was sub-cultured in defined liquid medium McVeigh-Morton culture medium at 35°C in a rotatory shaker(220 rpm) (Restrepo and Arango, 1980). After 5 or 7 days ofculture, the yeast cells were collected by centrifugation, thesupernatant was discarded, and the cells were washed threetimes in sterile PBS, pH 7.4. We determined the cell count ina haematocytometer and adjusted the inoculum suspensionto 3 ¥ 105 viable fungi per 50 mL. The cellular viability was90–95% in these experiments, as determined by vital stainingwith Evans blue (Sigma, St. Louis, MO, USA).

Intratracheal infectionTo mimic the most common infection in the chronic form ofPCM, BALB/c mice were inoculated intratracheally with P.brasiliensis Pb18, and after 30 days of infection, the animalswere subjected to a combined therapy of sulfamethoxazole/trimethoprim and a range of doses of P10 (1–40 mg) within

P10 entrapped within PLGA for mycosis treatmentAC Amaral et al 1127

British Journal of Pharmacology (2010) 159 1126–1132

PLGA. The animals were anaesthetized by intraperitonealinjection of 200 mL of a solution of 80 mg·kg-1 ketamine and10 mg·kg-1 xylazine (União Química, Brazil). After 10 min,their necks were hyperextended and incised to barely exposethe trachea. Each animal received 3 ¥ 105 viable fungi in 50 mLPBS using a 26.5-gauge needle. The incisions were suturedimmediately after the delivery of the fungi.

Antifungal treatmentThe infected mice were randomly divided into nine groups ofsix animals each and subjected to antifungal treatment withsulfamethoxazole/trimethoprim (15 mg·kg-1 and 3 mg·kg-1

respectively) in PBS, pH 7.4, with or without 20 mg P10 solu-bilized in 50 mL Freund’s adjuvant named ‘free’ or P10entrapped within PLGA at 1 mg, 5 mg, 10 mg, 20 mg or40 mg·50 mL-1. Group (i) was the control group treated onlywith PBS, group (ii) was treated with sulfamethoxazole/trimethoprim alone, group (iii) was treated withsulfamethoxazole/trimethoprim and 50 mL of ‘empty’ PLGAnanoparticles, group (iv) was treated with 20 mg·50 mL-1 of‘free’ P10, and groups (v) (vi) (vii) (viii) and (ix) were treatedwith 1 mg, 5 mg, 10 mg, 20 mg and 40 mg·50 mL-1, respectively,of P10 entrapped within PLGA. The treatment regimensstarted 30 days after fungal challenge and were continued for30 days. The empty PLGA nanoparticles given to group (iii)were prepared using the same procedure, methodology andratio amount of PLA : PGA (50:50) that was used to preparethe nanoparticles associated with P10, which gave the samefinal concentration in both preparations with or without P10.The particle size and size distribution were measured by laserlight scattering using a particle size analyser (Zetasizer,Malvern, UK). Size distribution was analysed over the range of1–1000 nm, and the mean diameter was calculated for eachsample. The PLGA particles with and without peptide P10 atvarious concentrations were measured, and the average diam-eter found was 430 � 5.1 nm for the loaded particle and 410� 4.9 nm for the empty one.

Animals from groups (ii) to (ix) received daily injections ofsulfamethoxazole/trimethoprim (15 mg·kg-1 and 3 mg·kg-1

respectively). Mice from groups (iv) to (ix) received four dosesof peptide P10 administered once a week during 4 weeks asfollows: 20 mg·50 mL-1 of ‘free’ or 1 mg, 5 mg, 10 mg, 20 mg and40 mg·50 mL-1 entrapped within PLGA. ‘Free’ P10 was admin-istered with complete Freund’s adjuvant in the hind paw, andP10 entrapped within PLGA at various concentrations wasadministered i.p. without adjuvant.

Fungal burden assayThe combined treatment effects were assessed by the residuallung, liver and spleen fungal burden. The animals from allexperimental groups were killed by cervical rupture 30 and 90days after the beginning of therapy, and the lungs, liver andspleen were aseptically removed and weighed. The organswere rinsed and homogenized in sterile PBS, pH 7.4, and100 mL of the homogenates was cultured in BHI agar supple-mented with 4% horse serum, 5% P. brasiliensis 192 culturefiltrate, 10 000 IU penicillin (Cultilab, Brazil) and 10 mg·L-1

streptomycin (Cultilab, Brazil) in duplicate. The Pb192

culture filtrate was prepared according to a previously pub-lished method (Singer-Vermes et al., 1992). The plates wereincubated at 37°C and colony-forming units (CFUs) werecounted 10 days post-plating to determine the CFU·g-1 oftissue.

Cytokine assaysTo assess cytokine production, we assayed IFN-g andinterleukins-4 (IL-4), -10 (IL-10) and -12 (IL-12) in lung homo-genates. Lung fragments were aseptically removed, weighedand homogenized in sterile PBS with protease inhibitor(Roche, USA). The cytokines were determined using commer-cial ELISA kits (BD Biosciences – Pharmingen, San Diego, CA,USA).

Statistical analysisStatistical Package for Social Sciences (SPSS) version 15 wasused to analyse our data. All results are expressed as means �

standard error. A one-way analysis of variance (ANOVA) withTukey’s post-test was applied to test inter-group differences.Differences between paired groups were analysed by theMann-Whitney test. P-values less than 0.05 were consideredsignificant.

MaterialsSulfamethoxazole was purchased from Sigma (St. Louis, MO,USA) and trimethoprim from Ducto (Bac-sulfitrin, Ducto).The poly-lactic acid (PLA) and poly-glycolic acid (PGA) usedto prepare the nanoparticles were purchased from Sigma (St.Louis, MO, USA). The P10 peptide was synthesized by the9-fluoroenylmethoxy-carbonyl technique (Huang et al., 1993)and provided by Dr Maria A. Juliano from the Department ofBiophysics of the Federal University of São Paulo. Drug andmolecular target nomenclature conforms to the BritishJournal of Pharmacology Guide to Receptors and Channels(Alexander et al., 2008).

Results

Fungal burden in treated animalsTo evaluate the efficacy of the combined therapy using theP10 peptide entrapped within PLGA in association withsulfamethoxazole/trimethoprim, we carried out in vivo anti-fungal drug experiments in BALB/c mice infected with thefungus P. brasiliensis, isolate Pb18. The treatment regimensstarted after 30 days of infection and were evaluated 30 and90 days after the beginning of treatment. The fungal load wasinvestigated in the lungs, liver and spleen of the animals. Nosignificant number of fungi in the liver and a very lownumber in the spleen were detected 30 days after intratracheal(i.t.) infection (data not shown). This result was expected asthe liver or/spleen infection is only detectable in the earlystages of this animal model of mycosis. In contrast, the mainfocus of the infection was the lungs. A high load of fungalcells was recovered in the lungs of the control animals thatreceived only PBS, both 30 and 90 days after beginning

P10 entrapped within PLGA for mycosis treatment1128 AC Amaral et al


the treatment (Figure 1). In the group treated only withsulfamethoxazole/trimethoprim, the infection was controlledduring the first 30 days of treatment followed by a highnumber of fungal cells after 90 days, comparable to theuntreated group (Figure 1).

A reduction of CFUs in comparison with untreated animalswas confirmed using 20 mg of ‘free’ P10 emulsified in Freund’sadjuvant (Marques et al., 2006), reversing the relapse ob-served in the group treated only with sulfamethoxazole/trimethoprim. The dose of 20 mg used for the ‘free’ form ofP10 peptide was chosen on the basis of earlier results (Tabordaet al., 1998; Taborda et al., 2004). Under this condition, lym-phoproliferative and cell-mediated immune responses pro-tecting mice from the P. brasiliensis infection were observed.In the groups subjected to the combined therapy ofsulfamethoxazole/trimethoprim and P10 entrapped withinPLGA, there was always a significant protection. After 30 daysof treatment, amounts in the range of 1–10 mg·50 mL-1 of P10encapsulated with PLGA were efficient as an adjuvant to che-motherapy, reducing the dose of the peptide necessary todecrease the fungal load and to avoid disease relapse by atleast 20-fold. The groups that received 20 or 40 mg·50 mL-1 ofP10 entrapped within PLGA, did not present a marked addi-tive action as adjuvants to chemotherapy (data not shown),although they responded better than the mice givensulfamethoxazole/trimethoprim alone. The PLGA nanopar-ticles without P10 showed a protective effect after 30 days,proving to be an adjuvant to chemotherapy by themselves,but these ‘empty’ nanoparticles were unable to prevent therelapse observed by sulfamethoxazole/trimethoprim treat-ment after 90 days of treatment. Thus,the fungal burdenrecovery was greater after treatment with empty nanoparticlesthan after any of the loaded nanoparticles of PLGA (contain-ing 1, 5 or 10 mg·50 mL-1 of P10 peptide; Figure 1).

Cytokine production induced by P10-PLGACytokine production was examined by monitoring the levelsof IFN-g, IL-4, IL-10 and IL-12 levels in lung tissue homoge-

nates from the animals given the combined therapy ofsulfamethoxazole/trimethoprim and peptide P10.

The data presented in Table 1 show the production of type1 cytokines IL-12 and IFN-g. The treatment with 20 mg of P10emulsified in Freund’s adjuvant or 1 mg of P10 entrappedwithin PLGA induced higher levels of IFN-g than in therespective controls after 30 and 90 days of the treatment.IFN-g is an important Th1 cytokine, which elicits protectionagainst P. brasiliensis infection. The levels of IL-12 were notsimilarly affected. Table 1 also shows changes in the cytokinesmore characteristic of Th2 responses (IL-4 and IL-10). After 30and 90 days of starting treatment, the group that receivedsulfamethoxazole/trimethoprim and 20 mg·50 mL-1 of ‘free’P10 emulsified in Freund’s adjuvant showed a significantdecrease of IL-10, thus confirming earlier results. P10entrapped within PLGA at 1 mg also reduced IL-10 after 30days and less after 90 days. Decrease of IL-10 with increasingP10 amounts in PLGA was less evident, and no decrease wasobserved in the levels of IL-4.

Discussion

In the present work, we examined the antifungal effects andthe protection elicited by a therapy using sulfamethoxazole/trimethoprim combined with the immunostimulatorypeptide P10 encapsulated into PLGA-dimercaptosuccinic acid(DMSA) for treatment of experimental PCM. The therapeuticefficacy and cytokine production were examined in a murinemodel of chronic PCM. Sulfamethoxazole/trimethoprim is alow-cost sulphonamide treatment that has been used in theinitial therapy of non-disseminated cases of PCM (Lortholaryet al., 1999; Travassos et al., 2008b) and it is recommended forlong-term treatments, but several cases of fungal resistanceand relapsing disease have been reported (Travassos et al.,2008a).

The development of immunotherapies using peptides asadjuvant molecules is a promising procedure that could be

70000

60000

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40000

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20000

10000

0

CF

U·g

lung

–1

PBS SULFA + TRIM PLGA/SULFA + TRIM

20 µg P10 “free”,SULFA + TRIM

1 µg P10-PLGA/SULFA + TRIM

5 µg P10-PLGA/SULFA + TRIM

10 µg P10·PLGA/SULFA + TRIM

30 days

90 days

*

##

#+*

Figure 1 Fungal burden recovery assessed by colony-forming units [CFU (g·lung·tissue)-1] in mice infected with P. brasiliensis Pb18 andsubjected to a combined therapy of sulfamethoxazole/trimethoprim (Sulfa + Trim; 15 mg·kg-1 and 3 mg·kg-1 respectively) and either20 mg·50 mL-1 P10 peptide solubilized in Freund’s adjuvant (‘free’) or P10 peptide (1, 5 or 10 mg·50 mL-1) entrapped within PLGA. Each barrepresents the average CFU (g·tissue)-1 with standard deviations. After 30 days of treatment, 1 mg·50 mL-1 of P10-PLGA/Sulfa + Trim yielded thebest response (lowest fungal CFU recovery) of all groups (+P < 0.05). After 90 days of treatment, no significant differences were found betweenthe responses of the ‘free’ P10 (20 mg·50 mL-1) and the P10-PLGA (5–10 mg·50 mL-1)-treated groups (marked with #). At day 90, a significantlylower number of fungal cells were recovered from mice treated with 1 mg·50 mL-1 P10-PLGA compared with the PLGA alone treated group(*P < 0.05). PLGA, poly(lactic acid-glycolic acid).



used to improve chemotherapy of fungal diseases. The useof P10 peptide has been effective in reducing the fungalburden in animals infected with P. brasiliensis, thus elicitingan efficient cellular immune response to combat thefungus (Taborda et al., 1998). The association of P10 withsulfamethoxazole/trimethoprim was effective in treating PCMand in avoiding the time-dependent relapse of the mycosis inthe murine model (Marques et al., 2006). The disadvantage ofthe use of the free peptide in vivo could be its lack of metabolicstability and the requirement for adjuvants not allowed forhuman use. A multiple antigenic peptide (MAP) constructcarrying four truncated P10 branches has been synthesizedand shown to be protective without additional adjuvants.However, the chemical synthesis of MAPs is a complex pro-cedure, and it is subject to errors during the process, makingtheir purification and characterization difficult (Taborda et al.,2004). Thus, efforts to prepare simpler and more efficientdelivery systems for immunostimulating peptides are stillneeded.

In the present work, PLGA-DMSA acid nanoparticlescontaining P10 in amounts ranging from 1 mg·50 mL-1 to40 mg·50 mL-1 were used combined with sulfamethoxazole/trimethoprim. The use of PLGA-based formulations for theencapsulation of these molecules aimed at reducing the fre-quency of injections and also protecting the peptide againstin vivo degradation while helping drug administration(Amaral et al., 2009). Nanoparticles composed of PLGA areappropriate devices for delivering the peptide due to the lowrate of co-polymer degradation and a constant release of thepeptide, which would be better at stimulating the immunecells. The proportion of PLA and PGA (50:50) used in this typeof drug delivery system presented a sustained release of theactive component over an average time of 72 h, as observed inother studies (Mittal et al., 2007; Amaral et al., 2009). Nano-particulated PLGA as a drug delivery system has been used forsome time based on its high stability, ready uptake into thecell by endocytosis and targeting of specific organs, withmany different mixtures of PLA and PGA (Dhiman et al.,

2000). There is no toxicity associated with this drug deliverysystem by itself and its use has been approved by drug regu-latory agencies, such as the Food and Drug Administration(Khang et al., 2003; Gabler et al., 2007). In vitro tests usingdifferent cell lines showed no toxicity for nanoparticulatedPLGA (Khandare and Minko, 2006; Gomes et al., 2008). PLGAby itself does have the advantage of eliciting cytotoxic T-cellresponses and also of inducing a mild inflammatory response,which could be involved in its adjuvant characteristics (Jianget al., 2005).

In our study, incorporating peptide P10 into PLGA reducedthe amount of this peptide necessary to decrease the fungalload in the infected animals and avoid disease relapse. A smallamount (1 mg·50 mL-1) of P10 in nanoparticles had the sameimmunotherapeutic effect as 20 mg·50 mL-1 of ‘free’ P10 emul-sified in Freund’s adjuvant over the first 30 days of treatment.However, the best protective effect of the PLGA-encapsulatedpeptide after 90 days of treatment was shown by the groupsthat received 5 or 10 mg·50 mL-1 of P10 in PLGA, making itpossible to reduce the amount of the peptide by at leastfourfold and preserving the protective capacity to avoidrelapse of the infection. Based on a previous report (Johansenet al., 2000), the entrapment of P10 within PLGA nanopar-ticles probably effectively protected it from in vivo enzymaticdestruction, which could explain the effectiveness of thelower doses of P10-PLGA.

Interaction of PLGA with antigen-presenting cells couldunderlie the improvement of immunomodulatory effects(Jaganathan et al., 2005). Further, the use of PLGA for con-trolled release of the peptide eliminates the need for an adju-vant, which becomes an advantage as only a limited numberof adjuvants are accepted for human administration (Jianget al., 2005). The protection elicited by 20 mg·50 mL-1 of ‘free’P10 in Freund’s adjuvant as well as P10-PLGA at 1 mg·50 mL-1

most probably depended on the induction of the high levelsof IFN-g, as noted in previous studies (Marques et al., 2006).Although the 10 mg P10-PLGA treatment showed low IFN-gand high IL-10 cytokine values after 90 days of therapy, it still

Table 1 Determination of cytokine production (pg·mL-1) on days 30 and 90 after starting treatment with sulfamethoxazole/trimethoprimand P10

Treatment Groups Th1 Th2

IL-12 IFN-g IL-4 IL-10

30 90 30 90 30 90 30 90

PBS 11.2 � 2.5 6.2 � 2.4 3.2 � 1.1 1.8 � 0.6 13.9 � 0.5 11.4 � 6.0 2.5 � 0.7 3.7 � 0.4#

Sulfa + Trim 7.3 � 0.7 6.4 � 0.1 2.3 � 0.3 2.5 � 0.1 11.4 � 2.6 11.2 � 0.3 4.1 � 0.2 3.7 � 0.2#

PLGA/Sulfa + Trim 6.7 � 0.7 6.8 � 2.0 1.3 � 0.4 1.8 � 0.2 10.7 � 0.4 8.6 � 0.2 2.8 � 0.9 3.2 � 0.4Sulfa + Trim/Freund/P10: 20 mg 10.9 � 1.0 6.1 � 1.9 7.5 � 1.0 10.6 � 2.4* 12.7 � 1.2 9.7 � 0.9 0.6 � 0.2 0.6 � 0.1Sulfa + Trim/PLGA/P10: 1 mg 7.0 � 1.9 5.5 � 2.7 5.1 � 0.8 7.7 � 1.1* 11.6 � 0.5 15.1 � 1.2 0.8 � 0.3 2.1 � 0.3Sulfa + Trim/PLGA/P10: 5 mg 8.4 � 2.5 8.7 � 3.5 1.0 � 0.1 2.9 � 1.2 17.9 � 6.7 12.1 � 2.2 2.0 � 0.8 2.0 � 0.5Sulfa + Trim/PLGA/P10: 10 mg 7.6 � 3.4 13.4 � 7.6 2.9 � 0.5 3.0 � 0.3 14.8 � 0.5 17.8 � 0.3 2.5 � 0.4 1.9 � 0.3#

*Not significant between groups, #different for 10 mg P10-PLGA compared with PBS and sulfamethoxazole/trimethoprim groups (P < 0.05).Values are means � standard deviations of measurements from each group.Bold: show high levels of IFN-g compared with the group treated with sulfamethoxazole/trimethoprim. These two groups also showed lower CFUs (see Figure 1).Italic: decrease in the production of IL-10 in the groups treated with free P10 and encapsulated P10 (1 mg) compared with the group treated with sulfamethoxazole/trimethoprim.CFUs, colony-forming units; IFN-gamma, interferon-g; IL, interleukin; PBS, phosphate-buffered saline; PLGA, poly(lactic acid-glycolic acid); Sulfa + Trim,sulfamethoxazole/trimethoprim.



managed to have a good protective effect. This may beexplained by the maintenance of high levels of IL-12 and lowproduction of IL-10 at 90 days, almost twofold lower thanthat produced in PBS and Sulfa + Trim-treated groups(Table 1), which could compensate for the low IFN-g levelnoted for this group. Most patients with the severe form ofPCM present a polarized Th2 response, with production ofIL-4 and IL-10 (Oliveira et al., 2002). In contrast, individualsexhibiting a polarized Th1 response, with production ofIFN-g, tend to resolve P. brasiliensis infections (Oliveira et al.,2002; Calich et al., 2008).

In conclusion, the use of PLGA as a carrier for peptide P10in combination with sulfamethoxazole/trimethoprim repre-sents a promising alternative to treat mycosis, as P10 elicits aTh1-like immune response able to control fungal infection.Sulfamethoxazole/trimethoprim is an inexpensive medicine,and its therapeutic efficacy can be increased by the cellularimmunity-stimulating P10 peptide. Encapsulation of peptideP10 within PLGA reduced the amount of this peptide neededfor a therapeutic response, up to 20-fold and avoided the useof an adjuvant. Additionally, in terms of application totherapy in humans, use of the PLGA nanoparticles shouldlead to improved antifungal protection and, at the same time,to a reduction in costs.

Acknowledgements

The authors wish to thank Conselho Nacional de Desenvolvi-mento Científico e Tecnológico (CNPq) for their financialsupport. Also, ACA was supported by a PhD fellowship fromCNPq.

Conflicts of interest

None to declare.

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DEGOBBI, C.; LOPES, F. D. T. Q. S.; CARVALHO-OLIVEIRA, R.; MUÑOZ, J. E.; SALDIVA, P. H. N. Correlation of fungi and endotoxin with PM2.5 and meteorological parameters in atmosphere of Sao Paulo, Brazil. Atmospheric Environment, v. 45, p. 2277-2283, 2011.

Correlation of fungi and endotoxin with PM2.5 and meteorologicalparameters in atmosphere of Sao Paulo, Brazil

Cristiane Degobbi a,*, Fernanda D.T.Q.S. Lopes b, Regiani Carvalho-Oliveira a,Julian Esteban Muñoz c, Paulo H.N. Saldiva a

a Laboratory of Experimental Air Pollution, Department of Pathology, Medical School, University of Sao Paulo, Av. Dr. Arnaldo 455, CEP 01246-903 Sao Paulo, SP, Brazilb Laboratory of Experimental Therapeutics 1, Department of Medical Clinic, Medical School, University of Sao Paulo, Av. Dr. Arnaldo 455, CEP 01246-903 Sao Paulo, SP, Brazilc Laboratory of Micology, Department of Microbiology, Institute of Biomedical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes, 1374 CEP 05508-900 Sao Paulo, SP, Brazil

a r t i c l e i n f o

Article history:Received 9 March 2010Received in revised form7 July 2010Accepted 1 December 2010

Keywords:FungiEndotoxinPM2.5Chemical composition

a b s t r a c t

Particulatematter, especially PM2.5, is associatedwith increasedmorbidity andmortality from respiratorydiseases. Studies that focus on the chemical composition of the material are frequent in the literature, butthose that characterize the biological fraction are rare. The objectives of this study were to characterizesamples collected in Sao Paulo, Brazil on the quantity of fungi and endotoxins associated with PM2.5,correlating with themass of particulatematter, chemical composition andmeteorological parameters. Wedid that by Principal Component Analysis (PCA) and multiple linear regressions. The results have shownthat fungi and endotoxins represent significant portion of PM2.5, reaching average concentrations of772.23 spores mg�1 of PM2.5 (SD: 400.37) and 5.52 EUmg�1 of PM2.5 (SD: 4.51 EUmg�1), respectively.Hyaline basidiospores, Cladosporium and total spore counts were correlated to factor Ba/Ca/Fe/Zn/K/Siof PM2.5 (p < 0.05). Genera Pen/Asp were correlated to the total mass of PM2.5 (p < 0.05) and colorlessascospores were correlated to humidity (p < 0.05). Endotoxin was positively correlated with the atmo-spheric temperature (p< 0.05). This study has shown that bioaerosol is present in considerable amounts inPM2.5 in the atmosphere of Sao Paulo, Brazil. Some fungi were correlated with soil particle resuspensionand mass of particulate matter. Therefore, the relative contribution of bioaerosol in PM2.5 should beconsidered in future studies aimed at evaluating the clinical impact of exposure to air pollution.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Particulate matter (PM) refers to a complex mixture of pollutantsconsistingof smoke, dust and all kindsof solid and liquidmaterial thatis in suspension in the atmosphere and it includes bioaerosols.There is a positive correlation between acute (Rivero et al., 2005) andchronic exposure (Churg et al., 2003; Popeet al., 2002) to atmosphericparticulate matter and risk of effects on health as airway inflamma-tion (Fujii et al., 2001; Peden, 2002), remodeling (Churg et al., 2003)and exacerbation of asthma (Peden, 2001; Peden, 2002), increasein cardiopulmonary diseases and cancer deaths, being the last twofactors related primarily to PM2.5 (Klemm et al., 2000; Pope et al.,2009, 2002; Schwartz et al., 1996). Previous studies conducted inSao Paulo also disclosed significant effects of ambient particles on

human health (Braga et al., 1999; Cendon et al., 2006) as well as inanimal models (Camargo Pires-Neto et al., 2006; Rivero et al., 2005).

There are many studies that characterize the chemical elementswhich account for such effects (Aust et al., 2002; Edgerton et al.,2006; Ostro et al., 2007; Turnbull and Harrison, 2000; Valliuset al., 2005; Wang et al., 2006), but fewer studies characterize thebiological components, such as fungi (Adhikari et al., 2006; Gliksonet al., 1995; Mastalerz et al., 1998) and bacteria components presentin the PM (Adhikari et al., 2006; Alexis et al., 2006;Mueller-Annelinget al., 2004), which are agents also related to respiratory illnesses,such as allergies and toxic responses (Alexis et al., 2003; Burge andRogers, 2000). The contribution of biogenic aerosols (bioaerosol) tothe health end-points related to ambient particle inhalation has notbeen fully understood. Bioerosol is composed by particles originatedby microbes, plant or animals, including living or dead organismsand their by products (Douwes et al., 2003). Fungal spores representa relevant part of the bioaerosol and are known risk factor foradverse health effects, such as inflammatory responses associated toallergies and asthma (Burge and Rogers, 2000; Bush and Portnoy,2001; Dales et al., 2003).

* Corresponding author. Tel.: þ55 (11) 3061 7254; fax: þ55 (11) 3064 2744.E-mail addresses: [email protected] (C. Degobbi), [email protected].

usp.br (F.D.T.Q.S. Lopes), [email protected] (R. Carvalho-Oliveira), [email protected] (J.E. Muñoz), [email protected] (P.H.N. Saldiva).

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Endotoxin, a component of the outer cell membrane of Gram-negative bacteria, may also be identified in ambient aerosols,mostly the fraction resulting from road dust (Salonen et al., 2004).Depending on inhaled doses and individual sensitivity, endotoxinmay elicit symptoms such as airway obstruction, asthma exacer-bation and pro-inflammatory cytokines (Kline et al., 1999).Bioaeosol can be adsorbed to particulate matter of other sources(traffic, industry, soil, for instance), having its aerodynamic andantigenic properties modified, enabling the penetration in deeperregions of the lung (D’Amato, 2002; Glikson et al., 1995; Knox et al.,1997). This topic is very important due to the fact that the particlesof biological origin may represent around 15% of the total partic-ulate (Matthias-Maser and Jaenicke, 1995) and from 5 to 10% of theresuspended particulate matter (Glikson et al., 1995).

Thus, the objectives of this study were to determine possiblecorrelations of these components of bioaerosol withmeteorologicalparameters, mass and chemical composition of PM2.5 and toestimate the relative contribution of endotoxins (components ofthe cell wall of gram-negative bacteria) and fungi in the PM2.5 inthe city of Sao Paulo, Brazil.

2. Methods

2.1. Collection and location of samples

The PM2.5 was collected from High-Vol sampler operating at1130 Lmin�1 (AVG-Energética Ltda.) coupled to an inlet (TischEnvironmental Inc, USA) that allows the separation of particles below2.5 mm (PM2.5). Some adjustments were made for the collection ofthree samples simultaneously for a 24 h period and the filters wereused for the characterization of fungi, endotoxins, and PM2.5.The samples were collected on the campus of the University of SaoPaulo, Brazil, located in an area of heavy traffic. The particles weresampled between April and July of 2008, totalizing 39 samples.

2.2. Characterization of fungi

Samples of fungi were collected at 1.2 Lmin�1 in MCE filters(Mixed cellulose ester filters, 0.8 um, 25 mm diameter, MilliporeBrazil) placed in cassettes of 25 mm (Millipore Brazil). This low flowrate allowed fungal spore identification under the microscopewithout visual interference of particle debris. After the collection,the filters were immediately removed from the cassettes anddissolved in Triacetin (C2: 0 e Cibraquim, Brazil) as described byYang and Heinsohn (2007). The slides were air dried, or quicklyheated until the filter was transparent. After this procedure, acidfucsin with lactic acid (Merck, Brazil) and slide covers were addedfor identification and counting on direct microscopy based onmorphology of the spores.

2.3. Endotoxins

Samples for analysis of endotoxin were collected at 10 Lmin�1

(Carty et al., 2003) in polycarbonate filters (0.4 mm, 37 mm diam-eter, Milipore Brazil). The filters were placed in the filter cassettesof 37 mm (Millipore Brazil). The storagewas performed by couplinga dissection chamber containing silica at the end of the filtermedium. The filters were stored at 4 �C until analysis. The extrac-tion was performed in 5 mL of Tween 20, 0.05% (Bioagency, Brazil)in pyrogen free water (Cambrex, Co) in endotoxin-free borosilicatetubes (Cambrex, Co). The tubes were placed in a sonicator for60 min at room temperature and the samples were vortexed every15 min. The suspensions were analyzed in duplicates using thekit Limulus Amebocyte Lysate (LAL) Pyrogent-5000 (Cambrex, Co).During plating, the Beta-blocker reagent (Cambrex, Co) was

added for inhibition of reaction with (1e3) b- D-glucan present inthe material, in concentration of 1:1. The reading was held inELx808LBS (Cambrex, Co) using the softwareWin KQCL. Escherichiacoli 055: B5 was used as standard.

2.4. Particulate matter

Teflon filters were used to collect PM2.5 (TefloTM W/Ring, PTFEMembrane W/PMP Ring, 2.0 ìm, 37 mm, Pall Corporation, Michi-gan, USA). The filters were weighed (balance UMX 2, Micronal SA)in an acclimatized room before and after collection to verify theamount of particulate matter in micrograms. Analysis of chemicalcomposition was done by the X-ray fluorescence EDX-700HS (Shi-madzu Corporation, Analytical Instruments Division, Japan) so thatthe results were issued in comparison to the standard NIST 2783.The chemical elements: Al, As, Ba, Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na,Ni, Pb, Sb, Ti, V, Zn, S, Si were subject to quantification in ppm.

2.5. Meteorological data

The data of temperature and relative humidity were provided bythe Department of Atmospheric Sciences USP/IAG/ACA, Campus ofthe University of Sao Paulo. Datawere collected in the daily averageat intervals of 5 min to be eventually converted to average dailydata. The meteorological station was located approximately 2 kmfrom the sampler.

2.6. Statistical analysis

It was used SPSS version 16.0. KolmogoroveSmirnov’s normalitytests were performed for the use of parametric or non parametrictests. Chemical elements were subjected to principal componentsanalysis (PCA) to be afterwards used in single and multiple linearregression involving fungi, endotoxins, PM2.5 and meteorologicalparameters. PCA was chosen as initial procedure because it allowsclustering of a high number of variables into small groups calledcomponents, which will explain the variability observed. The testswere considered significant to a < 0.05.

3. Results

3.1. Characterization of fungi

The fungal spores showed average values of 20,814 sporesm�3

(SD: 11,768 sporesm�3) and 772.23 spores mg�1 of PM2.5 (SD:400.37 spores mg�1 of PM2.5). It was possible to identify 22 types,the main ones listed in Table 1.

3.2. Principal components analysis

The PCA performed for chemical elements showed that threefactors were responsible for explaining 75% of the results:

Table 1Main types of fungal spores found from MCE filters. Mean values followed bystandard deviation.

Types of fungi (diameter< 2.5 mm) Concentration (Sporesm�3)a

Hyaline Basidiospores 14,153.83� 7,577.29Cladosporium sp 2,648.81� 2,523.26Penicillium/Aspergillus 727.75� 470.50Colorless ascospores 366.21� 678.65

Total amount 20,814.00� 11,768.00

a Mean and standard deviation.

C. Degobbi et al. / Atmospheric Environment 45 (2011) 2277e22832278

Factor 1 was composed of Ba, Ca, Fe, Zn, K and Si,Factor 2 was composed of Cr and Ni,Factor 3 was composed of Cu and S.

Concentration for each of the elements is shown in Table 2.

3.3. Fungi, PM2, 5 and meteorological data e multipleregression models

During the collection, it was found average concentrations ofPM2.5 of 32.85 m m�3 (SD: 14.87). The Multiple Linear Regressionmodel showed that hyaline basidiospores had positive correlationwith factor 1 of PM2.5 (R2¼ 0.184, p< 0.05) e Fig. 1a as wellCladosporium sp (R2¼ 0.406, p< 0.05) e Fig. 1b and total amount offungi (R2¼ 0.255, p< 0.05) e Fig. 1c. Neither of other parameters(i.e. temperature, humidity, PM2.5 mass and components 2 and 3)showed significance when adjusted in the model.

The genera Penicillium/Aspergillus (Pen/Asp) showed significantpositive correlation with the mass of the PM2.5 air (R2¼ 0.186,p< 0.05) e Fig. 2a. Neither of other parameters (i.e. temperature,humidity, and components1, 2 and 3) showed significance whenadjusted in the model.

Colorless ascospores showed significance when correlated tohumidity (R2: 0.16, p< 0.05) e Fig. 2b. Neither of other parameters(i.e. temperature, PM2.5 mass, and components1, 2 and 3) showedsignificance when adjusted in the model.

3.4. Endotoxins

It was possible to obtain results in twenty one samples withoutinterferences due to artifacts. The results for endotoxins rangedfrom 0.03 EUm�3 to 0.29 EUm�3 (mean 0.10 and SD: 0.07).The data for association with PM2.5 showed that the variation wasfrom 0.46 EUmg�1 PM2.5 to 15.81 EUmg�1 PM2.5 (mean 5.52 andSD: 4.51). Multiple linear regression models showed that endo-toxins were correlated only to the measures of temperature, withR2: 0.229, p < 0.05 (Fig. 2c). Neither of other parameters (i.e.humidity, PM2.5 mass and components1, 2 and 3) showed signifi-cance when adjusted in the model.

4. Discussion

Traditionally in the literature, there are efforts to quantify themaximum concentration of particulate matter to be inhaled daily orannually (CETESB, 2008; EPA, 1990). However, such an approachshows limitations since the elemental composition of the materialcan lead to different responses in the respiratory and cardiovascularsystems. Thus, recent studies aimed to characterize the particulatematter composition (Edgerton et al., 2006; Ostro et al., 2007; Wanget al., 2006), but surprisingly, few have focused in chemical andbiological composition, especially involving fungi characterization,even though these agents are consideredmajor sources of pollution,including in urban areas (Di Giorgio et al., 1996). Chemical elementsmay have a key role in the release of spores into the air, possiblycausing deleterious effects on metabolism, or, in contrast, favoringthe preferential adsorption of fungi to the particulate matter(Matthias-Maser et al., 1998). Furthermore, fungi may presentresistance to compounds such as heavy metals due to the differentphenotypes and genotypes between species (Gingell et al., 1976).

Although a lot of fungal spores have aerodynamics sizes biggerthan 2.5 mM (Burge and Rogers, 2000) and some studies have foundendotoxin in higher concentrations associated to PM10 (Heinrichet al., 2003), this particle diameter has been chosen becausestudies have demonstrated PM2.5 to be able of penetrating deepinto the lungs and it is more related to inflammatory responses andchanges in life expectancy than PM10 (Klemm et al., 2000; Popeet al., 2009; Schwartz et al., 1996). It is a matter of discussion ifbiological contaminants may act in synergy to contribute to sucheffects (Ning et al., 2000). To the best of our knowledge, there isno record about main genera provided by spore counts in theatmosphere of Sao Paulo, Brazil. Our study intent to supply initialdata in this field, because it is known that different generahave different allergens content and therefore, may trigger distincthealth responses after inhaled (Bush and Portnoy, 2001).

Endotoxin content has been chosen in detriment of culture-basedmethods orothermethodology, due todecrease inviability thatwouldoccur after sampling for period as long as 24 h, media selectivity ofsome species and because endotoxin is recognized as a potentinflammatory agent present in live or dead cells (Douwes et al., 2003).

Our estimations have shown that the fungi are a significantportion of the PM2.5. Still, the chemical composition seems tobe correlated to the increase of spores in the atmosphere. The sporecounts of hyaline basidiospores and Cladosporium spp werecorrelated to factor 1 of PM2.5, which corresponds to elements thatsignalize traffic of vehicles and crustal resuspension (Amato et al.,2009; Schauer et al., 2006). The low humidity associated withheavy traffic may be responsible for a high aerolization of the crustalelements due to resuspension of road dust, thus increasing therelease of bioaerosol from soil, vegetation (Salonen et al., 2004). Thefact that bioaerosol is incorporated in the same factor as elementsmarkers of traffic poses a new challenge when interpreting therole of automotive emissions on health. Silicon was identified as anelement associated to increased pulmonary inflammation in rodentsinhaling concentrated ambient particles (Saldiva et al., 2002).The inflammatory reaction associated to silicon inhalation could notbe explained by changes in their toxic potential by the particleconcentrator (Savage et al., 2003). Since fungal spores may accountfor these kinds of health effects (Cooley et al., 2000; Kauffman et al.,2000; Young et al., 2001) and were not measured in both studies, itis plausible to speculate that bioaerosol combined with crustalelements resuspension may play a role in the aforementionedfindings. Indeed, in places near the streets of heavy traffic it may befound a higher concentration of spores in suspension (Lugauskaset al., 2003). A study made in Brisbane, Australia took measure-ments of bioaerosols (ie fungi and pollen) in the atmosphere

Table 2Elementary characterization of PM2.5.

Element (ngm�3) Concentrationa

Al 407.15� 307.21As 4.14� 0.85Ba 8.15� 6.15Ca 209.53� 161.05Co 3.13� 0.03Cr 14.52� 9.01Cu 75.12� 34.46Fe 443.94� 314.50K 731.87� 326.19Mg 70.87� 31.33Mn 56.09� 20.75Na 1287.96� 351.07Ni 30.42� 6.46Pb 35.46� 40.78Sb 2.24� 1.48Ti 26.63� 23.35V 13.04� 7.89Zn 198.25� 146.28S 699.32� 463.01Si 649.58� 412.13

a Mean and standard deviation.

C. Degobbi et al. / Atmospheric Environment 45 (2011) 2277e2283 2279

associated with the particulate matter. It was verified the cytos-plasmatic material of fungi adsorbed in the material from vehicularsources. The authors have not discarded the possibility of mecha-nisms of synergy between both (Glikson et al., 1995). Later, the samegroup found that the fungi were the most abundant bioaerosolsin the atmosphere and again, cytosplasmatic material associatedwith particles from vehicle exhaust and resuspension in the crust(Mastalerz et al., 1998). These results are in accordance with ourresults, suggesting that the particulatematter can affect fungi, whichare then subject to be more abundant in the atmosphere due to theturbulencemechanisms (resuspension of elements in the crust) andcomposition (elements of combustion or heavy traffic).

Cladosporium spp. is a typical fungus from organic and surfacesof leaves material (Awad, 2005; Levetin and Dorsey, 2006) andmechanisms of resuspension of the crust may be responsible forincreasing the amount of such spores in the air. Studies whichcompare rural areas with urban ones have correlated this fungus tourban development (Awad, 2005). Other data show that this fungusmay be relatively tolerant to heavy metals such as Zinc, Cadmiumand Lead, being Zinc one of the elements of Factor 1 (Gingell et al.,1976).

In the case of genera Pen/Asp, the models have shown that thevariation of these fungi may be better explained by the amountof particulate matter in the atmosphere. These results agree withthose found in the atmosphere of Taipei in which it was also foundamoderate correlation between PM10 and the genus Penicillium (Linand Li, 2000). A study made in three regions of Egypt (two rural andtwo urban areas) found greater number of genera Aspergillus andPenicillium in urbanized regions (Awad, 2005). Although the authorshave suggested that these results could have been due to transportfrom rural areas, these data help to sustain the results of this study.Nevertheless, colorless ascospores show positive correlation withhumidity. These results were expected, since these spores are pro-tected in asci during periods of drought, only to be airborne duringperiods of moisture (Troutt and Levetin, 2001). Typically, samplescollected after heavy rains showa tendency to have a high amount ofthese elements in the air (Haines et al., 2000).

4.1. Endotoxins

The results have shown that endotoxins ranged from 0.03 to0.29 EUm�3 (mean 0.10 and SD: 0.07). These results are superior

A B

C

Fig. 1. Multiple Linear Regression for fungi and Factor 1 (p< 0.05). a. Hyaline basidiospores. b. Cladosporium sp. c. Total amount of fungi.


to those found by other authors, in the atmosphere of Munich,Germany, with average values of 0.015 EUm�3 (Carty et al., 2003).One comparative study among Danish cities have shown that trafficjammed streets may have higher concentrations of endotoxins thanin rural areas, with median values of 4.4 and 2.9 EUm�3, respec-tively. In town areas, the values found are close to those found inthis study, with median values of 0.33 EUm�3 (Madsen, 2006).

In relation to the amount of endotoxin associated with thePM2.5 the concentrations ranged from 0.46 to 15.81 EUmg�1 ofPM2.5 (average of 5.52 EUmg�1 of PM2.5). For the study conductedin Munich by Carty and collaborators (2003), the average concen-trationwas 1.07 EUmg�1 of PM2.5. Another German study found anaverage of 1.2 EUmg�1 of PM2.5 (Heinrich et al., 2003). In some U.S.cities, it was demonstrated an average concentration in the externalenvironment of 2.0 EUmg�1 of PM2.5 (Long et al., 2001). In otherregions such as Boston, MA, it was found an average of 2.3 EUmg�1

of PM2.5 in ambient of ambient particle concentrator (Imrichet al., 2000). Our results are shown to be below those collected inthe southeast of Mexico City, with an average of 12.22 EUmg�1

of PM2.5 (Osornio-Vargas et al., 2003). Although our results haveshown similarity with others in literature, it is quite difficult to

make comparisons, due to diversity of sampling and analyticalmethods.

The regression analysis have shown that endotoxin was corre-lated only with increasing temperature, partly agreeing with otherstudies in which the greatest amount of endotoxin was foundduring the warm seasons and correlated positively with increasingtemperature and decreased moisture (Carty et al., 2003). In otherEuropean cities, larger amounts of endotoxinwere also found in thewarmer seasons of the year (Madsen, 2006). Further studies oncharacterization of the culturable bacteria have also correlatedthe concentration of bacteria in the air to temperature increases(Di Giorgio et al., 1996).

5. Conclusions

Our results have shown that bioaerosol (i.e. fungal spores andendotoxin) is present in considerable amounts in the fine particlemode in the atmosphere of Sao Paulo. Some of bioaerosol compo-nents, mainly fungi, are associated to soil resuspension and massof particulate matter. Endotoxin seems to be more influenced bychanges in temperature. Future studies regarding clinical analysis

A B

C

Fig. 2. Multiple Linear Regression for fungi and endotoxin p < 0.05. a. Penicillium/Aspergillus and mass of PM2.5. b. Colorless ascospores and relative humidity. c. Endotoxin andatmospheric temperature.


of the impacts of concurrent exposure to these agents are needed toverify possible modulatory or synergistic effects between biologicaland chemical constituents of PM2.5.

Acknowledgments

We thank the FAPESP (Fundação de Amparo à Pesquisa doEstado de São Paulo) for funding this research.

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ROSSI, D. C. P.; MUÑOZ, J. E.; BELMONTE, R.; CARVALHO, D. D.; FAINTUCH, B.; BORELLI, P.; MIRANDA, A.; TABORDA, C. P.; DAFFRE, S. Therapeutic use of a cationic antimicrobial peptide from the spider Acanthoscurria gomesiana in the control of experimental candidiasis. BMC Microbiology, v. 12, p. 1-12, 2012.

RESEARCH ARTICLE Open Access

Therapeutic use of a cationic antimicrobialpeptide from the spider Acanthoscurriagomesiana in the control of experimentalcandidiasisDiego C Rossi1, Julian E Muñoz2, Danielle D Carvalho3, Rodrigo Belmonte1, Bluma Faintuch4, Primavera Borelli5,Antonio Miranda6, Carlos P Taborda2 and Sirlei Daffre1*

Abstract

Background: Antimicrobial peptides are present in animals, plants and microorganisms and play a fundamentalrole in the innate immune response. Gomesin is a cationic antimicrobial peptide purified from haemocytes of thespider Acanthoscurria gomesiana. It has a broad-spectrum of activity against bacteria, fungi, protozoa and tumourcells. Candida albicans is a commensal yeast that is part of the human microbiota. However, inimmunocompromised patients, this fungus may cause skin, mucosal or systemic infections. The typical treatmentfor this mycosis comprises three major categories of antifungal drugs: polyenes, azoles and echinocandins;however cases of resistance to these drugs are frequently reported. With the emergence of microorganisms thatare resistant to conventional antibiotics, the development of alternative treatments for candidiasis is important. Inthis study, we evaluate the efficacy of gomesin treatment on disseminated and vaginal candidiasis as well as itstoxicity and biodistribution.

Results: Treatment with gomesin effectively reduced Candida albicans in the kidneys, spleen, liver and vagina ofinfected mice. The biodistribution of gomesin labelled with technetium-99 m showed that the peptide is capturedin the kidneys, spleen and liver. Enhanced production of TNF-a, IFN-g and IL-6 was detected in infected micetreated with gomesin, suggesting an immunomodulatory activity. Moreover, immunosuppressed and C. albicans-infected mice showed an increase in survival after treatment with gomesin and fluconazole. Systemicadministration of gomesin was also not toxic to the mice

Conclusions: Gomesin proved to be effective against experimental Candida albicans infection. It can be used as analternative therapy for candidiasis, either alone or in combination with fluconazole. Gomesin’s mechanism is notfully understood, but we hypothesise that the peptide acts through the permeabilisation of the yeast membraneleading to death and/or releasing the yeast antigens that trigger the host immune response against infection.Therefore, data presented in this study reinforces the potential of gomesin as a therapeutic antifungal agent inboth humans and animals.

BackgroundAntimicrobial peptides (AMPs) are components of theinnate immune system of vertebrates and invertebrates,having a broad-spectrum activity against bacteria, fungi,viruses and protozoa [1]. In general, AMPs are small

molecules with 1 to 10 kDa of molecular mass and exhi-bit a high content of basic amino acids, which results inan overall positive net charge. AMPs also usually havean amphipathic structure. Thus, while the positivecharges of basic amino acids facilitate interaction withthe negative charges of the phospholipids of biologicalmembranes, the hydrophobic amino acids facilitate theinsertion of AMPs into the membrane, which will even-tually lead to lysis of the microorganisms. Some AMPs

* Correspondence: [email protected] of Parasitology, Institute of Biomedical Sciences, University ofSão Paulo, Av. Prof Lineu Prestes, 1374, 05508-900 São Paulo, SP, BrazilFull list of author information is available at the end of the article

Rossi et al. BMC Microbiology 2012, 12:28http://www.biomedcentral.com/1471-2180/12/28

© 2012 Rossi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.


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can act on internal targets, such as the inhibition ofnucleic acid and/or protein synthesis [1,2]. Alternatively,some AMPs selectively boost the host immune responsethrough the regulation of the production of proinflam-matory cytokines and chemokines and by promoting thechemotaxis of T cells, monocytes, neutrophils and eosi-nophils. They can also effect dendritic cell differentia-tion and stimulate angiogenesis [3].Gomesin is a cationic AMP isolated from haemocytes

of the tarantula spider Acanthoscurria gomesiana[4].This peptide contains 18 amino acids and two disul-phide bridges and adopts a b-hairpin structure [5]. Thedisulphide bridges provide stability in mammalianserum and resistance to proteolysis [6]. Gomesin exertsa strong microbicidal activity against Gram-positive andGram-negative bacteria, filamentous fungi, yeast, para-sites and tumour cells through a mechanism of pore for-mation or “detergent like” action [4,7-9].Candidiasis is an infection caused by fungi from the

genus Candida and can affect the skin, eyes, oral cavity,oesophagus, gastrointestinal tract, vagina and vascularsystem of humans. Most infections occur in patientswho are immunocompromised or debilitated [10]. Vul-vovaginal candidiasis is the most common form ofmucosal disease, affecting up to 75% of women (reviewby [11]). In Brazil, candidiasis has become a publichealth problem. It is the 3rd leading cause of death fromsystemic mycosis in AIDS-negative patients. Recordsindicate an increase in mortality from an annual averageof 39 deaths between 1996 and 1998 to 54 between2005 and 2006. Taking in account the deaths of AIDSpatients with underlying cases of candidiasis, the diseaseis the 2nd leading cause of death from systemic mycosis,with 1,780 deaths in Brazil from 1996 to 2006 [12].Nosocomial candidiasis is also a public problem in Bra-zil [13]. In the USA, Candida species are the fourthleading cause of nosocomial bloodstream infections inseveral hospitals and the mortality from 1997 to 2003was approximately 0.4 deaths per 100,000 populationper year (review by [14,15]). The leading treatment ofCandida infections is done with polyenes (amphotericinand liposomal amphotericin), azoles (fluconazole andvoriconazole) and echinocandins (caspofungine) [16].Regardless of which antifungal drug is used, there is fre-quent treatment failure [16]. In this paper, we show thepotential therapeutic use of gomesin in an experimentalinfection of C. albicans.

ResultsEvaluation of the antifungal activity of gomesin in vitroThe minimum inhibitory concentration (MIC) of gome-sin in the isolate 78 and strain ATCC 90028 was 5.5μM and 11 μM, respectively, while the MIC of Flucona-zole in the isolate 78 and strain ATCC 90028 was 186

μM and > 1.5 mM, respectively. In addition, weobserved growth inhibition of the isolate 78 with thecombined treatment of 0.6 μM gomesin and 3.5 μM flu-conazole. Growth inhibition of strain ATCC 90028 wasobserved with the combined concentration of 1.3 μMgomesin and 14.3 μM fluconazole (Table 1). Further-more, the fractional inhibitory concentration index(FICI) of the combination of gomesin and fluconazolewas 0.11 in isolate 78 and 0.19 in strain ATCC 90028(Table 1).

Evaluation of the antifungal activity of gomesin in micewith disseminated and vaginal candidiasisTreatment with 5 mg/kg and 15 mg/kg of gomesin inmice with disseminated candidiasis effectively reducedthe fungal burden of the kidneys, spleen and liver whencompared with the control group (PBS-treated mice)(Figure 1A-C). Treatment with 10 mg/kg and 20 mg/kgof fluconazole also effectively controlled the infection(Figure 1A-C). Moreover, treatment of vaginal candidia-sis with 0.2% and 0.5% gomesin and 2% miconazoleshowed a significant decrease in colony forming units(CFUs) when compared with vehicle treatment (controlgroup) (Figure 1D). The combination of gomesin andfluconazole or miconazole did not result in a synergisticeffect.

Cytokine levels in kidneys of gomesin-treated miceTreatment with gomesin and fluconazole significantlyincreased the concentration of TNF-a, IFN-g and IL-6in the kidneys compared to controls that were notinfected and not treated as well as controls that wereinfected and treated with PBS (Figure 2).

Evaluation of the effect of antifungal drugs inimmunosuppressed mice with disseminated candidiasisThe group of infected animals that received PBS (con-trol) reached 100% mortality on the fifteenth day afterinfection. No statistically significant difference wasobserved between the group treated with gomesin (5mg/kg) and the group treated with fluconazole (20 mg/kg), although there was an increase in survival duringthe last treatment. Nonetheless, the combined treatmentof 5 mg/kg of gomesin and 20 mg/kg of fluconazoleproduced a survival rate of 23% within 30 days afterinfection, which was statistically significant. The controlgroups that were not infected or those that receivedPBS or 5 mg/kg of gomesin remained alive until the endof the experiment (Figure 3).

In vivo toxicityGomesin administration did not alter the number ofleukocytes in the non-infected mice. However, whenspecific cell populations were analysed, the number of


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neutrophils and eosinophils were increased, whereas thenumber of lymphocytes was decreased. The administra-tion of gomesin did not alter the haemoglobin levels.Nevertheless, treatment with gomesin resulted in anincrease in the percentage of circulating reticulocytes.Moreover, the administration of gomesin showed nochange in the levels of total bilirubin, direct and indir-ect, as well as creatinine and gamma-GT (Table 2).

Biodistribution of radiolabeled gomesinThe biodistribution of gomesin labelled with techne-tium-99 m was evaluated in the kidneys, spleen andliver (Figure 4). The liver had the highest percentage ofradiolabeled peptide detected (60%), which persisted forup to 24 h post-injection, whereas the kidneys showed aradioactive peak at 120 min followed by a gradualdecrease during the following hours. The spleen was the

Table 1 Minimum inhibitory concentration (MIC) and fractional inhibitory concentration index (FICI) of gomesin andfluconazole

MIC (μM) FICI

C. albicans (78) C. albicans(ATCC 90028)

C. albicans (78) C. albicans(ATCC 90028)

Gomesin 5.5 11 - -

Fluconazole * 186 - -

Gomesin + Fluconazole 0.6 + 3.5 1.3 + 14.3 0.11 0.19

* = not detected in up to 1.5 mM

Figure 1 Gomesin treatment of mice infected with C. albicans. Evaluation of the number of colony forming units (CFU) per gram of tissue ofthe kidneys (A), spleen (B), liver (C) and vagina (D). The disseminated candidiasis was performed by intravenous injection of 3 × 105 yeastssuspended in 100 μL of PBS and vaginal candidiasis was performed by inoculating 3 × 106 yeasts suspended in 20 μL of PBS. The treatment wasdone one, three and six days after infection with C. albicans (strain 78). Animals were treated with different doses of gomesin (GOM), fluconazole(FLUCO) and miconazole (MICO). As a control, infected animals received only PBS or cream (CREAM). * Indicates statistical significance (ANOVAwith post-Tukey test, P < 0.05).


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lowest of the organs tested (less than 5% detected) andwas stable for only 60 min after administration of tech-netium-99 m-labelled gomesin, dropping to undetectablelevels after 120 min.

DiscussionGomesin is an antimicrobial peptide isolated from hae-mocytes of the spider Acanthoscurria gomesiana andhas a broad-spectrum of activity against bacteria, fungi,protozoa and tumour cells [4,7,9,17,18]. The antifungalactivity of gomesin in vitro has previously been reported[4,7]. However, the antifungal activity against clinical

Figure 2 Cytokine levels in kidneys . Cytokine levels wereevaluated in the kidneys of mice treated with gomesin (5 mg/kg)and fluconazole (20 mg/kg). Non-infected and untreated animals(NINF), as well as infected animals that received PBS, were used ascontrols. * Indicates statistical significance (t-test, P < 0.05)compared to the control INF.

Figure 3 Survival of immunosuppressed mice withdisseminated candidiasis treated with antifungal drugs. Animalswere treated with 100 mg/kg of cyclophosphamide and infectedwith 103 yeasts of C. albicans (INF). The animals were treated with 5mg/kg of gomesin (GOM), 20 mg/kg of fluconazole (FLUCO) or thecombination of 5 mg/kg gomesin and 20 mg/kg of fluconazole. Ascontrols, infected animals (NINF) received PBS and uninfectedanimals received PBS and gomesin 5 mg/kg. * Indicates statisticalsignificance (Long-rank test, P < 0.05).

Table 2 Evaluation of the toxicity of the gomesintreatment

NINF* NINF + GOM**

Leukocytes (mm3) 4637 ± 1114 4462 ± 1580

Neutrophils (mm3) 846 ± 288 1208 ± 388***

Eosinophils (mm3) 46 ± 46 135 ± 72***

Lymphocytes (mm3) 3744 ± 981 2660 ± 437***

Hemoglobin (g/dL) 13 ± 0.9 13 ± 0.5

Reticulocytes (%) 5.5 ± 0.7 9.3 ± 2.8***

Total Bilirubin (mg/dL) 0.48 ± 0.23 0.3 ± 0.1

Direct bilirubin (mg/dL) 0.35 ± 0.19 0.2 ± 0.1

Indirect bilirrubin (mg/dL) 0.13 ± 0.13 0.09 ± 0.009

Creatinine (mg/dL) 0.32 ± 0.09 0.34 ± 0.05

Gamma-GT (mg/dL) < 1 U/L < 1 U/L

* Non-infected mice

** Non-infected mice treated with gomesin (GOM)

*** p < 0.05


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isolates of Candida albicans resistant to antifungaldrugs has not been studied. In this paper, we analysedthe antifungal activity of gomesin in vitro and in vivoagainst a clinical strain of C. albicans (isolate 78), aswell as its biodistribution and toxicity in mice.Our data showed that C. albicans (isolate 78) is resis-

tant to fluconazole up to 1.5 mM, but gomesin is effec-tive against this strain at a lower concentration (MIC =5.5 μM). This resistance to fluconazole is a commoncause of treatment failure [19]. A synergism betweengomesin and fluconazole against two isolates of Candidaalbicans (78 and ATCC 90028) was demonstrated usingthe FICI calculation method. The synergistic mechanismof gomesin and fluconazole is not completely under-stood, but studies with Cryptococcus neoformanssuggested that gomesin, through membrane permeabili-sation, promotes an increased entry of fluconazole intothe fungal cytoplasm, which results in a better inhibitionof the ergosterol synthesis. In this way, fluconazole iseffective against C. neoformans at lower doses whenapplied in combination with gomesin [7]. A similar phe-nomenon was observed in murine melanoma cells(B16F10-Nex2) treated with gomesin and the monoclo-nal Mab A4M in vitro. The cytotoxicity of Mab A4Mwas only detected in the presence of gomesin, after per-meabilisation of the cell membrane allowed the entryand action of the monoclonal antibody [9]. From thesestudies, we hypothesised that gomesin facilitates theentry of fluconazole in Candida albicans through mem-brane permeabilisation.The literature on the use of antimicrobial peptides in

the treatment of disseminated candidiasis is ratherscarce. A study of the HLF peptide (1-11) originatedfrom lactoferrin in immunosuppressed mice with

disseminated candidiasis showed that a single dose of0.4 ng/kg, 24 h after infection, was able to significantlyreduce CFU in the kidneys [20]. ETD-151, an analogueof heliomicin also has been shown to be particularlyeffective against systemic candidiasis in comparison withamphotericin B and several azoles [21]. Likewise, treat-ment with gomesin proved to be effective against disse-minated candidiasis. The peptide effectively reduced thefungal burden in the kidneys, which is the highest trop-ism organ for Candida. A similar effect was observedwith fluconazole; however, this drug has some toxiceffects and has selected resistance in Candida albicans[19]. Therefore, the use of gomesin as a therapeutic maybe an alternative treatment for candidiasis because ourresults show that it is non-toxic in mice. Unlike in vitrotreatment with gomesin and fluconazole, we have notdetected any the synergistic effect of treatment withboth drugs in vivo.The treatment and prevention of recurrent vaginal

candidiasis includes the use of imidazoles and triazolesas a first-line treatment, unless it is caused by a con-firmed or suspected azole-resistant Candida strain. Theefficacy of both oral and local therapy is similar, but, thelocal treatment presents several advantages, including areduction of adverse effects; however, local treatment iscontraindicated during pregnancy and breast feeding[22]. In recent years, there has been a focus on bothunderstanding drug resistance to antifungal agents andoptimising therapy of Candida infections [23]. Thereare no reports of topical treatment with antimicrobialpeptides against vaginal candidiasis. In this paper, weare the first to describe an effective topical formulationof an antimicrobial peptide that is able to reduce CFUscount in an experimental vaginal candidiasis model. Wefound that 0.2% and 0.5% gomesin cream reduced theCFU on vaginas of the animals by 10 fold when com-pared to control animals. Minor changes in the treat-ment protocol with gomesin, either by increasing thefrequency or changing the doses, may potentially pro-duce better results. Treatment with 2% miconazolecream was also effective in controlling the CFUs of thevaginas of the animals. However, it was necessary to usea dose of miconazole that was at least four times higherthan the dose of gomesin to produce a similar effect. Nosynergistic effect was observed after treatment with acombination of gomesin and miconazole.In addition to the direct action of AMPs on microor-

ganisms, either through membrane permeabilisation orinternal target interference [2], it has been reported thatsome AMPs may possess an immunomodulatory func-tion [3]. In order to verify if gomesin has such activity,the concentrations of IFN-g, TNF-a and IL-6 were eval-uated in the kidneys of mice that had been infected withC. albicans and treated with this peptide. These

Figure 4 Biodistribution of gomesin. After administration ofradiolabeled gomesin (99mTc-HYNIC-gomesin), the liver, kidneysand spleen were dissected at different time points to assess thebiodistribution of the peptide.


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cytokines, especially IL-6, activate neutrophils, whichplay an essential role in the defence mechanism againstCandida[24]. We observed that treatment with 5 mg/kggomesin significantly increased the concentration of thethree cytokines analysed. A similar effect was alsoobserved with fluconazole treatment. The increase ofcytokine levels in the kidneys might help to control can-didiasis through the activation of the host immune sys-tem. This action appears to be similar to that observedwith another AMP, murine b defensin-2, which acts viaTLR4 and leads to the production of various cytokines,such as IL-12 and IL-6, as well as chemokines [25].However, we cannot dismiss the hypothesis that thedirect action of gomesin can trigger the release ofpathogen-associated molecular patterns, or PAMPs,which would exacerbate the immune response of ani-mals. This has been previously reported for the antimi-crobial peptide human b defensin-2 [26]. The use ofantimicrobial peptides as immunomodulatory agents fortherapeutic application is an effervescent field in pro-gress [27].After verifying that the gomesin treatment was effec-

tive against disseminated candidiasis in healthy mice, wedecided to evaluate the activity of gomesin in immuno-suppressed animals, as candidiasis is typically observedin immunocompromised hosts [10]. Treatment withgomesin (5 mg/kg) showed no significant increase insurvival compared to control animals. This suggests thatthe direct action of gomesin was not sufficient to con-trol the infection and that immunomodulatory action isrequired to suppress the candidiasis. Treatment with flu-conazole (20 mg/kg) also did not result in a significantincrease in the survival of treated animals as comparedto control animals. However, the combined treatment of5 mg/kg gomesin and 20 mg/kg of fluconazole resultedin 23% survival of mice 30 days after infection. Thiscould be due to gomesin facilitating the entry of fluco-nazole into the yeast, thus leading to the survival of ani-mals. Another hypothesis is that treatment withfluconazole, being fungistatic, would allow time forgomesin to act.To evaluate whether gomesin could be used as a ther-

apeutic treatment for C. albicans infection, we per-formed blood analyses to determine the toxicity ofgomesin in mice. No difference in the total number ofleukocytes was observed in animals treated with gome-sin. However, the number of eosinophils in mice notinfected with Candida albicans but treated with gome-sin was higher than the control group. The eosinophiliacaused by gomesin may be due to the induction of anallergic response. Further experiments are needed inorder to evaluate this effect. We have also noticed thatgomesin treatment leads to a higher number of

neutrophils. This effect might be a consequence of theinduction of the pro-inflammatory response by gomesin,which would stimulate the bone marrow to recruit neu-trophils. However it is not currently known if these cellsare being recruited to the site of infection.In addition, gomesin did not change the haemoglobin

levels, which suggests that this peptide was not toxic toerythrocytes. However, the quantity of reticulocytes isgreater in treated animals, suggesting that the peptideprovokes an erythropoiesis compared to control animals(non-gomesin treated). Perhaps treatment with gomesincauses hypoxia in animals, thus increasing erythropoietin[28]. Furthermore, gomesin was not nephrotoxic or hepa-totoxic, as the bilirubin, creatinine, and Gamma GT levelsfrom treated animals are similar to the control group.Therefore, gomesin seems to be non-toxic to mice.In addition to the evaluation of toxicity, the biodistri-

bution of gomesin was performed to understand itspharmacokinetics and therefore its therapeutic potential.The biodistribution data revealed that the peptidemainly accumulates in the liver, although it also accu-mulates in the kidneys and spleen, within the first sev-eral minutes after administration. This suggests a rapidclearance from the circulation. The presence of gomesinin the sites of infection might explain the reduction ofCandida albicans observed in our experiments. How-ever, other studies are needed to monitor the excretionof the peptide.

ConclusionsGomesin was effective against Candida albicans infec-tion in vitro and in vivo. Gomesin can be used as analternative treatment for candidiasis, either alone or incombination with fluconazole. Although the mechanismof action of gomesin is not fully understood, it has beensuggested that it directly acts on the fungal membraneand/or stimulates the immune response against yeastinfection. Data presented in this study reinforces thepotential of gomesin as a therapeutic antifungal agent inboth humans and animals.

MethodsAntimicrobial compoundsThe chemically synthesised gomesin was obtained fromGENEPEP (France) with 97% purity analysed by liquidchromatography - mass spectrometry. Fluconazole wasobtained from Pfizer (Pfizer Inc., New York) and mico-nazole from Janssen Pharmaceutica (Janssen-Pharma-ceutica, Beerse). Gomesin and fluconazole weredissolved in PBS for the in vivo assays and water for invitro tests. Miconazole was dissolved in PBS with 20%dimethyl sulfoxide (DMSO) for incorporation into thevaginal cream.


Page 6 of 9

Candida albicans strainsTwo strains of Candida albicans were used: isolate 78[29] and the isolate ATCC 90028. Periodically, isolate 78was inoculated into mice in order to maintain itsvirulence.

In vitro studiesThe antifungal activity of antimicrobial compounds wasevaluated by using the protocol M-27A2, according tothe Clinical and Laboratory Standards Institute (CLSI)[30]. Briefly, 80 μl of RPMI 1640 with 1.6 M MOPS pH7 containing 104 yeast/mL of C. albicans in logarithmicgrowth phase, were added to the wells of a polypropy-lene 96-well plate containing 20 μl of serial two-folddilution of gomesin (starting at 44 μM), fluconazole(starting at 1,488 μM) or the combination of gomesin(starting at 11 μM) and fluconazole (starting at 115μM). After 48 h of incubation at 37°C fungal growthwas evaluated by determining the absorbance at 595nm. The lowest concentration that inhibited 100%growth was considered the minimum inhibitory concen-tration (MIC). The fractional inhibitory concentrationindex (FICI) was determined following the methodologydescribed previously [31].

AnimalsBALB/c mice (6- to 8-week-old males or females) werebred at the Animal Facility at the Institute of BiomedicalScience of University of São Paulo, Department ofImmunology under specific pathogen-free conditions.Food and water were given ad libitum. All animals werehandled in accordance with good animal practice asdefined by the relevant national animal welfare bodiesand all in vivo testing was approved by the InstitutionalAnimal Care and Use Committee of the University ofSão Paulo, reference number: 87/42. For immunosup-pression of animals, doses of 100 mg/kg cyclophospha-mide were administered intraperitoneally 4 days and 1day before infection with C. albicans, the third day afterinfection and, from this point on, every 4 days until theend of treatment [32]. The animals were kept in cageslined with wood shavings and closed with autoclaved fil-ter, and served autoclaved food and water in order tomaintain a sterile environment. Cages were exchangedtwice a week in laminar flow hoods. The animals wereconsidered anergic when the number of leukocytes wasfound to be less than 100 cells/mm3 [33]. The vaginalcandidiasis model was developed by inducing the pseudooestrus phase by the subcutaneous administration of 0.5mg of 17 beta-estradiol valerate (Sigma Chemicals, StLouis), dissolved in sesame oil (Sigma Chemicals, StLouis) 3 days before the vagina’s infection [34]. Swissmice were provided by the Animal Facility of IPEN-CNEN for the biodistribution studies.

InfectionsOne colony of C. albicans (isolate 78) was selected fromthe plate dishes and incubated in brain heart infusion(Oxoid, England) at 37°C for 24 h with 200 rpm agita-tion. The sediment obtained by centrifugation at 1500 gfor 5 min was washed three times in PBS and resus-pended in 5 mL of PBS. The number of yeast per mL ofthis suspension was determined with a Neubauerchamber.The disseminated candidiasis was induced by intrave-

nous injection of 3 × 105/100 μL of PBS and the immu-nosuppressed model was induced by intravenousinjection of 103 yeasts suspended in 100 μL of PBS.Vaginal candidiasis was induced by inoculating 3 × 106

yeasts suspended in 20 μL of PBS.

In vivo treatmentsMice with disseminated candidiasis were treated withgomesin and fluconazole. The drugs were administeredintraperitoneally in a final volume of 500 μl at the fol-lowing concentrations: gomesin (2.5 mg/kg, 5 mg/kgand 15 mg/kg), fluconazole (10 mg/kg and 20 mg/kg)and a combination of both (2.5 mg/kg to 5 mg/kggomesin and 10 mg/kg to 20 mg/kg fluconazole). Formice with vaginal candidiasis, gomesin (0.02%, 0.2% and0.5%), miconazole (2%) and a combination of both (0.2%gomesin and 2% miconazole) were incorporated into avaginal cream (10% Wax self-nonionic emulsifier, 2%mineral oil, 5% propylene glycol and 84% distilled water,pH 4.5) for topical application. For all treatments, drugswere administered 1, 3 and 6 days after infection withC. albicans. An equivalent volume of PBS or cream wasadministered to the control animals. To evaluate thefungal burden, the kidneys, spleen, liver and vagina ofthe mice were dissected aseptically on the seventh dayafter infection, weighed and homogenised in 1 mL ofPBS. Aliquots of the homogenate (100 μl) were inocu-lated on brain and heart infusion (Oxoid, England) con-taining 2% agar. After incubation for 18 h at 37°C, thenumber of CFUs was determined. The effectiveness oftreatment was determined by comparing the number ofCFUs per gram of tissue of treated animals with thenumber of CFUs per gram of tissue of control animals(untreated). For the survival curves, the animals weremonitored daily for 30 days.

Measurement of cytokinesThe kidneys of animals infected with C. albicans andtreated with gomesin or fluconazole were dissected,washed in PBS and homogenised with an electric tissuehomogeniser in 1 mL of PBS containing a cocktail ofprotease inhibitors (Sigma Chemicals, St Louis). Thiscocktail of protease inhibitors was composed of 1 μg/mlof pepstatin A (aspartyl protease inhibitor), 4 mM


Page 7 of 9

benzamidine (seine protease inhibitor), 1 mM ethylene-diaminetetraacetic acid acetic (metallo-protease inhibi-tor) and 1 mM N-Ethylmaleimide (cysteine proteaseinhibitor). Non-infected animals or animals infected andnot treated were used as controls. The concentrations ofIFN-g, TNF-a and IL-6 were evaluated on a flow cyt-ometer (BD FACSCaliburTM, San José) using the kitCytometric Bead Array and Mouse Inflammation™ (BD,San José) and the methodology described by themanufacturer.

Blood analysisBlood was collected by puncturing the brachial plexus ofanesthetised mice using EDTA (1%) as an anticoagulantafter 7 days of gomesin administration (15 mg/kg). Reti-culocytes cells and leukocytes were counted by standardmethods. The haemoglobin concentration was deter-mined using the modified Drabkin method. Blood sam-ples were prepared on microscopic glasses, dried andstained with May-Grünwald reagents for morphologicalexamination of the blood. The number of reticulocyteswas determined in blood smears stained with SupraVital New Methylene Blue. We also determined thelevels of bilirubin, creatinine and gamma GT biochemi-cally using the Sims-Horm, Enzyme and Alkaline picratemethods, respectively.

Evaluation of the biodistribution of radiolabelled gomesinwith technetium-99 m in miceHYNIC-gomesin was manually synthesised by solidphase methodology as described previously, exceptthat pyroglutamic acid was substituted for 6-hydrazinonicotinamide (HYNIC) [6]. The HYNIC-gomesin con-jugate was labelled with the radioisotope technetium-99 m obtained from an alumina-based 99Mo/99mTcgenerator, supplied by the Radiopharmacy Centre ofthe Institute of Energetic and Nuclear Research(IPEN/CNEN). Briefly, 20 mg of tricine and 5 mg ofethylenediamine N,N’-diacetic acid (EDDA) were dis-solved in 0.5 ml of 0.1 M PBS, previously nitroge-nated. Ten micrograms of HYNIC-gomesin, 5 μl of 8.9mM SnCl2·2H2O solution in 0.1 N HCl (nitrogen-purged) and 500 μl of Na99mTcO4 was added to thevial. The reaction was conducted by heating the solu-tion at 100°C for 20 min in a water bath and thenallowing it to cool to room temperature. The pH ofthe reaction mixture was 7 [35]. The product 99mTc-HYNIC-gomesin (0.1 mL), with an approximate activ-ity of 74 MBq (2 mCi), was administered to the tailvein of the mice. The animals were sacrificed in aCO2 chamber at 5, 30, 60, 120, 240, 360, and 1,440min after injection of the radiolabeled gomesin. Sixanimals were used for each time point. The kidneys,spleen and liver of each animal was dissected and

transferred to tubes to measure radioactivity. Theradioactivity count was performed in the GammaCounter Shaft type NaI, using the same standard dosein injected animals minus the radioactivity retained inthe site injection (tail). The uptake of radiolabeledgomesin by each organ was calculated using the fol-lowing equation: DI% = (CPM organ/standard CPM) ×100), where%DI = percentage of the injected dose andCPM = count per min [35].

Statistical analysisANOVA, with the post-Tukey test, was used to evaluatethe statistical significance of results obtained in allexperiments except the blood and survival analysis,where the Students t-test and Log-rank test were used,respectively. The differences between the resultsobtained with treatment compared to the controls wereconsidered statistically significant when the p value wasless than 0.05.

AcknowledgementsWe are grateful to Susana P. Lima for technical assistance and CassianoPereira for figure preparation. This work was supported by Brazilian grants:Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and theConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Author details1Department of Parasitology, Institute of Biomedical Sciences, University ofSão Paulo, Av. Prof Lineu Prestes, 1374, 05508-900 São Paulo, SP, Brazil.2Department of Microbiology, Institute of Biomedical Sciences andLaboratory of Medical Mycology IMT/SP - LIM53, University of São Paulo, SãoPaulo, SP, Brazil. 3Department of Special Analysis, SD&W Modelagem eSoluções Estratégicas Ltda., São Paulo, SP, Brazil. 4Radiopharmacy Center,Institute of Energetic and Nuclear Research, São Paulo, Brazil. 5Department ofClinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, SãoPaulo University, São Paulo, SP, Brazil. 6Departament of Biophysics, UniversityFederal of São Paulo, São Paulo, SP, Brazil.

Authors’ contributionsDR carry out in vitro, in vivo studies and measurement of cytokines,participated in biodistribution experiments, blood analysis, experimentaldesign and helped to write the manuscript. JEM participated in the in vivo,in vitro studies and measurement of cytokines. RB participated in in vivo andin vitro studies. AM synthetised the HYNIC-gomesin. BF was responsible forthe biodistribution experiments. PB carries out the blood analysis. DDC wasin charge of the statistical analysis. CPT and SD conceived of the study andmanuscript preparation. All authors read and approved the final manuscript.

Received: 5 September 2011 Accepted: 6 March 2012Published: 6 March 2012

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doi:10.1186/1471-2180-12-28Cite this article as: Rossi et al.: Therapeutic use of a cationicantimicrobial peptide from the spider Acanthoscurria gomesiana in thecontrol of experimental candidiasis. BMC Microbiology 2012 12:28.

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RITTNER, G. M. G.; MUÑOZ, J. E.; MARQUES, A. F.; NOSANCHUK, J. D.; TABORDA, C. P.; TRAVASSOS, L. R. Therapeutic DNA Vaccine Encoding Peptide P10 against Experimental Paracoccidioidomycosis. Plos Neglected Tropical Diseases, v. 6, p. e1519-9, 2012.

Therapeutic DNA Vaccine Encoding Peptide P10 againstExperimental ParacoccidioidomycosisGlauce M. G. Rittner , Julia1 ´ n E. Munoz1, Alexandre F. Marques , Joshua D. Nosanchuk , Carlos P.1 2

Taborda1,3, Luiz R. Travassos4*

1 Institute of Biomedical Sciences, Department of Microbiology, University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil, 2 Departments of Medicine, and Microbiology and

Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 3 Medical Mycology Laboratory-IMTSP and LIM53/HCFMUSP, University of

Sao Paulo, Sao Paulo, Sao Paulo, Brazil, 4 Cell Biology Division, Department of Microbiology, Immunology and Parasitology, Federal University of Sao Paulo (UNIFESP), Sao

Paulo, Sao Paulo, Brazil

Abstract

Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in SouthAmerica. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM isresponsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericinB or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response inmice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, whichsuggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmidimmunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection withP. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungalburden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architectureand eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunizationwith these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective ofbeing also effective in human patients.

Citation: Rittner GMG, Munoz JE, Marques AF, Nosanchuk JD, Taborda CP, et al. (2012) Therapeutic DNA Vaccine Encoding Peptide P10 against ExperimentalParacoccidioidomycosis. PLoS Negl Trop Dis 6(2): e1519. doi:10.1371/journal.pntd.0001519

Editor: Jeffrey Michael Bethony, George Washington University, United States of America

Received April 8, 2011; Accepted December 23, 2011; Published February 2 , 2012

Copyright: � 2012 Rittner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: CPT and LRT were supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (Fapesp) grants 09/15823-7, 07/07588-2, 06/50634-2, 05/02776-0; National Research Council, CNPq, 470513/2009-8. CPT and LRT are research fellows of CNPq. The funders had no role in study design, data collection andanalysis, decision to publish,or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Paracoccidioides brasiliensis is a thermally dimorphic fungus that

causes a systemic granulomatous disease known as paracoccidioi-

domycosis (PCM). PCM is widespread in Latin America, mainly

affecting rural workers, and its incidence has increased in recently

deforested areas associated with soil churning [1]. Acquisition of P.

brasiliensis may arise from inhalation of aerosolized conidia.

Recently we reviewed the death rates by systemic mycoses in

Brazil [2]. PCM was the principal cause of death identified for

3,583 patients in the 1996–2006 decade and representing

51.2% of total deaths due to systemic mycoses. It ranks as the

10th most common cause of death among infectious diseases in

Brazil [2].

There are distinct forms of PCM. The acute and sub-acute

forms affect both genders with primary involvement of the

reticuloendothelial/lymphatic system. The chronic form affects

mainly adult males and predominantly causes pulmonary and/or

mucocutaneous disease [3]. Antifungal chemotherapy is required

for treatment, though treatment may not assure complete

eradication of the fungus, with frequent relapses. Treatment with

itraconazole usually takes 6–9 months in the low and 12–18

months in the moderately severe cases. Frequently, a combination

of trimethoprim and sulfamethoxazole (TMP/SMZ) is used, held

for 12 months in the low severity forms and 18–24 months in the

moderately severe forms. Patients with severe PCM forms require

endovenous treatment with anfothericin B or the TMP/SMZ

association for long periods, monitored by clinical, radiological

and serological tests [4].

The 43 kDa glycoprotein was characterized as the major

diagnostic antigen of P. brasiliensis [5,6,7]. Immunization with gp43

elicited delayed hypersensitivity reactions in guinea pigs [8] and

humans [9], implying the presence of T-CD4+ reacting epitopes.

Based on the sequence of gp43 [6], which encodes a polypeptide of

416 amino acids with a single high mannose N-glycosylated chain

[10], the T-cell epitope was mapped to a 15-mer peptide called

P10 [11]. The hexapeptide HTLAIR comprises the essential core

of P10 that induces proliferation of lymph node cells from mice

sensitized to gp43 or infected with P. brasiliensis [11]. Type 1-T

helper lymphocytes producing IL-2 and IFN-c are induced by P10

[11,12,13]. Intratracheally infected mice previously immunized

with P10 in the presence of complete Freund’s adjuvant (CFA) had

.200-fold reduction of lung P. brasiliensis colony-forming units

(CFUs). In many cases the immunization rendered preserved lung

www.plosntds.org 1 February 2012 | Volume 6 | Issue 2 | e1519

8

architecture with few or no yeasts, whereas the infected,

unimmunized mice displayed dense pulmonary inflammation

characterized by epithelioid granulomas with numerous yeast cells

[11,12].

The immunoprotection by P10 depends on the IFN-c-

producing Th-1 response since mice deficient in IFN-c, IFN-c-R

or IRF-1, but not IFN-a-R/IFN-b-R, were not protected by P10

immunization [12] The essential role of IFN-c in organizing

granulomas that contain P. brasiliensis yeasts has also been

recognized by other investigators [13,14,15].

Several experimental avenues have been pursued to validate

P10 as a vaccine candidate. These studies have included: a) the

presentation of P10 by MHC molecules from different murine

haplotypes [11]; b) its conservation in nature, confirmed by

examining gp43 molecules from different isolates [16]; c) its

immunogenicity and effective immunoprotection in formulations

that do not require complete Freund’s adjuvant [17]; d) its

presentation by most human HLA-DR molecules as well as that of

neighbor peptides to P10, based on the sequence of gp43 [18]; and

e) the effectiveness of P10 as an adjuvant to chemotherapy in

normal [19] and anergic [20] mice challenged intratracheally with

virulent P. brasiliensis.

The immunoprotective properties of P10 emulsified in Freund’s

adjuvant have been well documented in an established murine

model of PCM [11]. Since CFA is not allowed in human vaccines

and a tetramer of truncated P10 although immunogenic, involves

laborious chemical methods [17], we have explored alternative

approaches for P10 delivery. In the present work we have

investigated the effectiveness of plasmid immunization with P10

and/or IL-12 inserts given prior to or after challenge with a

virulent Pb18 isolate of P. brasiliensis using a murine pulmonary

PCM disease model. Our results demonstrate that plasmid

immunization with P10 with or without IL-12 inserts is highly

therapeutic in mice intratracheally infected with this fungus. Most

importantly, immunization was effective either prior to, or after

infection suggesting that these plasmids are candidates for use in

human PCM.

Materials and Methods

Plasmid constructionsYeast cells of P. brasiliensis isolate 18 (Pb18) were grown in

Sabouraud Dextrose Broth (BD, MD, USA) at 37uC for 7 days.

Cells were washed and frozen in liquid nitrogen then disrupted by

grinding on a mortar. Total RNA was isolated with Trizol

according to manufacturer’s instruction (Invitrogen, CA, USA).

Complementary DNA was synthesized from 1 mg of total RNA in

the presence of oligo(dT)18 (Fermentas, MD, USA) and Revertaid

M-MuLV(Fermentas, MD, USA).

The P10 nucleotide sequence was obtained using the sense

PCR primer derived from the gp43 [6] 59 nucleotide sequence:

[59-AAT AAG CTT CAA ACC CTG ATC GCC-39], and the

antisense primer derived from the 39 end of the gp43 gene: [59-

AAT GAA TTC ATT GGC GTA ACG GAT TGC-39]. A

HINDIII site and an EcoRI site were added to the sense and

antisense primers, respectively, for cloning into plasmid pcDNA3

(Invitrogen, CA, USA). PCR reactions (50 ml) were carried out

following the protocol provided by Fermentas, using 100 ng of

cDNA and 100 ng of each primer. The P10 PCR reaction started

with one cycle at 94uC (2 min), followed by 40 cycles at 94uC(30 sec), 55uC (1 min) and 72uC (1 min), and a final 7-min

extension at 72uC. PCR products were purified using Wisard SV

gel and PCR Clean-UP system (Promega, Brazil) and each PCR

product was digested with the appropriate restriction enzyme

(Fermentas) and cloned into the pcDNA3 by directional insertion

in the HINDIII/EcoRI sites. The resulting plasmid was called

pP10.

Plasmid pORF-mIL-12 was acquired from InvivoGen (CA,

USA). The confirmation of the insert was done using the primers:

sense [59-CGG GTT TGC CGC CAG AAC ACA-39] and

antisense [59-GGC CAC CAG CAT GCC CTT GT-39]. The IL-

12 PCR started with one cycle at 94uC (2 min), followed by 40

cycles at 94uC (1 min), 45uC (1 min) and 72uC (2 min), and a final

7-min extension at 72uC.

Preparation of plasmid DNATo prepare plasmid DNA for immunization, Escherichia coli

XL1Blue and DH5a cells were transformed by electroporation

using Cellject Duo according to the manufacturer’s directions

(Hybaid, Middx, UK) with the DNA constructs or the vector

plasmid alone and then cultured at 37uC in Luria broth

supplemented with ampicillin (50 mg/ml).

The positive clones were confirmed by automatic sequencing

carried out following the protocol provided by Applied Biosystems

(CA, USA) and analyzed by BioEdit and Blast. The parental

vectors, pcDNA3 and pORF were used as negative controls. DNA

for immunization was purified using the EndoFree Giga Kit

(Qiagen, CA, USA) and was diluted in TE buffer to the final

concentration of 1 mg/ml.

Plasmid gene expression in mammalian cellsFor the expression of pORF-mIL-12 in mammalian cells, a

transient-transfection assay was performed using Lipofectin

(Invitrogen) and 1 or 2 mg plasmid transfected into HeLa cells

(26105 cells/well). The cells were grown in RPMI medium

supplemented with 10% fetal calf serum (FCS) (Cultilab, SP,

Brazil). After 24 h incubation, the cells were harvested, and total

RNA was isolated with Trizol for reverse transcription (RT)-PCR.

IL-12 PCR was used as described above. IL-12p70 was detected

(80 ng/ml) by ELISA, in the supernatant of transfected HeLa

cells.

Author Summary

Paracoccidioidomycosis (PCM) is the predominant system-ic mycosis in Latin America causing half of the total deathsamong systemic fungal infectious diseases in Brazil.Chemotherapy is the standard treatment, but the longtime required, severe cases of immunosuppression andfrequent relapses indicate that additional methods shouldbe introduced such as immunotherapy combined withantifungal drugs. Previously, the protective activity of P10,a peptide derived from the major diagnostic antigen gp43,was demonstrated, alone or combined with chemothera-py. P10 elicited a vigorous IFN-c mediated Th-1 immuneresponse. Presently, the reduction of fungal load, and evensterilization, was attempted using a specific DNA vaccineencoding P10. Plasmid pcDNA3 expression vector with P10insert was tested as a vaccine in intratracheally infectedBALB/c and B10.A mice. Our results showed that vaccina-tion with pP10 induced a significant reduction of thefungal burden in the lung. Co-vaccination of pP10 with aplasmid encoding mouse IL-12 proved to be even moreeffective in the elimination of the fungus with virtualsterilization in a long term infection and treatment assaysystem. The data suggest that immunization with theseplasmids, without the need of an adjuvant, could be usedin the prevention and treatment of PCM in humanpatients.

Experimental DNA Vaccine against PCM


P10 expression from vector pcDNA3, followed by IFN-cproduction

DNA immunization was performed by injecting groups of 5 six-

week-old male BALB/c mice intramuscularly in both quadriceps

with three doses of 100 mg of plasmid encoding P10 (pP10), 50 mg

of either pP10 and pcDNA3 vector alone, or 50 mg of the pcDNA3

vector alone, each in 50 ml of TE buffer. A total of three

immunizations were given at weekly intervals in alternating sites

on the left and right hind legs. The mice were euthanized one

week after the last immunization, their spleens were isolated and

single-cell suspensions were prepared by gentle homogenization in

RPMI medium supplemented with 1% FCS. Cells were suspended

and treated with isotonic ammonium chloride to lyse erythrocytes.

The splenocytes were washed by centrifugation, suspended in

RPMI containing 10% FCS, and dispensed into wells on a

microtitering plate (56105 mononuclear cells per well). The

cultures were stimulated with 20 mg/ml of synthetic P10. After 24

and 48-h incubation at 37uC with 5% CO2, supernatants were

collected and IFN-c was assayed by a sandwich enzyme-linked

immunoassay (ELISA) (BD Pharmingen, CA, USA). Splenocytes

from animals immunized with pP10 and stimulated with synthetic

P10 produced 10 and 15 ng/ml IFN-c after 24 and 48 h,

Figure 1. Summary of the treatment protocols used. First protocol: BALB/c mice received 4 weekly injections. Animals were infected andsacrificed 30 or 60 days later. Second protocol: BALB/c and B10.A mice were infected intratracheally. Mice received 4 weekly vaccine doses andanimals were sacrificed 1 week after the last injection. Third protocol: B10.A mice were infected i.t. and one month after infection, they wereimmunized with the DNA vaccine. The animals were sacrificed one month after the last injection, six months after infection.doi:10.1371/journal.pntd.0001519.g001



respectively. When 50 mg of pcDNA3 was used for immunization,

9 and 11 ng/ml IFN-c was released by splenocytes at the two

examined times.

Ethics statementThis study was carried out as recommended by the Brazilian

college of animal experimentation (COBEA). The protocol has

been approved by the Ethical Committee on Animal Experimen-

tation of University of Sao Paulo (Permit number: 039).

AnimalsBALB/c and B10.A mice were bred at the Institute of

Biomedical Science of University of Sao Paulo, Department

of Immunology animal facility under specific-pathogen-free

conditions.

Fungal strainYeast cells of the virulent isolate Pb 18 of P. brasiliensis were

maintained by weekly subculturing on Sabouraud Dextrose Agar

and incubation at 37uC. Before experimental infection, 7–10 day-

old cells were inoculated into Sabouraud Dextrose Broth and

incubated at 37uC for 5–7days with rotary shaking. Fungal cells

were washed three times in phosphate-buffered saline pH 7.2

(PBS) and counted in a haemocytometer. The viability of fungal

cells in the inoculum was determined by staining with Janus B

(Merck, Darmstadt, Germany) and was greater than 90%.

Intratracheal infection of BALB/c and B10.A miceBALB/c and B10.A mice (6- to 8-week-old males) were

inoculated intratracheally (IT) with 50 ml suspension of 36105

Pb18 yeast cells in sterile saline (0.85% NaCl). Mice were

anesthetized i.p. with 200 mL of a solution containing 80 mg/kg

ketamine and 10 mg/kg of xylazine (both from Uniao Quımica

Farmaceutica, Brazil). After approximately 10 min, their necks

were extended to expose the trachea at the thyroid level and cell

suspensions were injected with a 26-gauge needle. The incisions

were sutured with 5-0 silk.

Immunization of mice with plasmid DNAThree different protocols were used (Fig. 1). Injections of 50 mg

plasmid were given in the quadriceps muscle. First protocol:Groups of 10 BALB/c mice were injected with PBS (control),

pcDNA3 (50 mg; control), pORF (50 ug; control), plasmid

encoding P10 (pP10, 50 mg), plasmid encoding IL-12 (pIL-12,

50 mg) or with both pP10 and pIL-12 (50 ug each). A total of 4

injections were given weekly on alternating sites, on the left and

right hind legs. One week after the last injection, mice were

infected intratracheally then sacrificed 30 or 60 days later.

Second protocol: BALB/c and B10.A mice (10 mice per group)

were infected intratracheally. One month after infection, the mice

received 4 weekly injections of either PBS (control), pcDNA3

(50 mg; control), pP10 (50 mg), pIL-12 (50 mg) or both pP10 and

pPIL-12 (50 ug each). Mice were sacrificed 1 week after the last

Figure 2. Immunoprophylactic treatment of PCM. Gene immunization was initiated 30 days before fungal challenge. CFUs are from lungs ofBALB/c mice infected intratracheally with 36105 yeast cells and subjected to immunization with vectors containing P10 (pP10) and/or IL-12 (pIL-12)DNA insert. Control mice were inoculated with PBS or with vectors without insert. Mice were sacrificed 30 (&) and 60 (%) days after infection. Eachbar represents the average counts and standard deviations of CFUs in lungs from 10 animals in each group. Experiments were carried out in triplicatewith similar results. **p#0.0001, comparing vector with and without insert; ## p#0.0001, .comparing untreated and other groups.doi:10.1371/journal.pntd.0001519.g002



injection. Third protocol: B10.A mice (10 mice per group) were

infected intratracheally. One month after infection, they were

treated with 4 weekly injections followed by a monthly booster for

3 additional months (total of 7 injections). The injections were with

either PBS (control), pcDNA3, pP10, pIL-12 or both pP10 and

pPIL-12. The mice were sacrificed one month after the last

injection, six months after infection.

Fungal burden in organs of infected miceMice were sacrificed and the lungs, liver and spleen were

removed. Weighed tissue sections were homogenized and then

washed 3 times with PBS and suspended in 1 ml PBS. Suspensions

(100 ml) were inoculated on brain-heart infusion (BHI) agar medium

supplemented with 4% FCS and 5% spent culture medium of P.

brasiliensis (strain-192), streptomycin/penicillin 10 IU/ml (Cultilab)

and cycloheximide 500 mg/ml (Sigma, MO, USA). Colonies were

counted after 10 days of incubation at 37uC.

HistopathologyLung sections from sacrificed mice were fixed in 10% buffered

formalin for 24 h and embedded in paraffin. Four-micra sections

were stained with haematoxylin-eosin (HE) or silver nitrate

(Gomori) and examined microscopically (Optiphot-2; Nikon,

Tokyo, Japan).

Cytokine detectionSections of excised lungs were homogenized in 2 ml of PBS in

the presence of protease inhibitors: benzamidine HCl (4 mM),

EDTA disodium salt (1 mM), N-ethylmaleimide (1 mM) and

Pepstatin (1.5 mM) (Sigma, St Louis, MO). The supernatants were

assayed for IL-4, IL-10, IL-12, and IFN-c using ELISA kits (BD

OpTeia, San Diego, CA). The detection limits of the assays were

as follows: 7.8 pg/ml for IL-4, 31.3 pg/ml for IFN-c and IL-10,

62.5 pg/ml for IL-12, as previously determined by the manufac-

turer.

Statistical analysisStatistical analyses were performed using GraphPad Prism5

software. The results are expressed as means and standard

deviations (SD). The nonparametric Kruskall-Wallis honestly

significant difference test was used. p values are shown in the

Figure legends.

Figure 3. Therapeutic treatment of PCM. Gene immunization started 30 days after infection. CFUs are from lungs of BALB/c (%) and B10.A(&)mice infected intratracheally with 36105 yeast cells and subjected to immunization with vectors containing P10 (pP10) or IL-12 (pIL-12) DNA inserts.Control mice were inoculated with PBS or with vectors without insert. Mice were sacrificed 60 days after infection. Each bar represents the averagecounts and standard deviations of CFU in lungs from 10 animals in each group. Experiments were performed three times and similar results wereachieved. *p#0.05, **p#0.005, comparing vector with and without insert; ## p#0.005, comparing untreated and other groups.doi:10.1371/journal.pntd.0001519.g003



Results

Colony-forming units (CFU) in mice immunized prior toinfection (prophylactic immunization). First protocol

To explore the effects of the plasmid with the P10 insert (pP10)

with or without the murine IL-12 gene insert (pIL-12), BALB/c

mice were immunized and then infected intratracheally with

virulent P. brasiliensis Pb 18. Animals were sacrificed after 30 or 60

days, and the fungal burden in the lungs, spleens and livers was

determined. The number of lung CFU per gram of tissue was

significantly reduced in animals immunized with pP10 and/or

pIL-12 compared to controls at both time intervals (Fig. 2).

Notably, we observed that the empty plasmids (pcDNA3 and

pORF) also induced a significant reduction in CFU relative to

mice that received PBS alone, which is presumably a result of

dendritic cell activation through Toll-like receptor 9 binding of

plasmid unmethylated CpG motifs. Immunostimulation by DNA

from P. brasiliensis also attributed to CpG motifs showed protective

effects in susceptible mice [21,22]. Nevertheless, the fungal load

measured in CFUs in mice receiving pP10 and/or pIL-12 was

significantly lower than that in mice treated with control pcDNA3

and pORF. Livers and spleens from all animals had no detectable

fungal cells.

Organ CFUs in mice immunized 1-month after infection(therapeutic immunization). Second protocol

The therapeutic protocol attempts to reproduce the clinical

reality of patients presenting to medical attention after developing

symptomatic PCM. We studied two mouse strains with different

susceptibilities to PCM, BALB/c (susceptible) and B10.A (highly

susceptible) [23]. The data showed that immunization with pP10

and/or pIL-12 was therapeutic in mice infected with P. brasiliensis

for 1 month prior to receiving plasmid immunizations (Fig. 3).

CFU reductions were significant in infected mice receiving pP10

and/or pIL-12 compared to mice injected with PBS or pcDNA3.

In contrast to the first protocol, injection of pcDNA3 after

installing PCM was not sufficient to reduce the fungal burden. The

most significant reduction in the lung CFUs from B10.A mice was

achieved when pP10 and pIL-12 were combined. The CFUs from

the livers and spleens were barely detectable in all groups.

Organ CFUs in a long-term infection model of B10.A micesubmitted to gene immunization. Third protocol

This protocol allowed us to analyze the efficacy of therapeutic

plasmid treatment during long-term infection (six months) of the

highly susceptible mouse strain, B10.A. Treatment of mice with

PCM using pP10 and/or pIL-12 significantly reduced lung CFUs

(Fig. 4). However, the impact of pIL-12 alone was not as dramatic

as either pP10 alone or pP10 with pIL-12. Notably, treatment with

the combination of pP10 and pIL-12 virtually eradicated the

infection in all organs examined.

Lung histopathologyThe lungs of control animals in each experimental protocol

group showed intense inflammation and large numbers of yeast

cells, whereas mice receiving pP10 with or without pIL-12 had

significantly reduced inflammation, and lower or undetectable

fungal cells. Analysis of the lungs of animals from the third

protocol (6 months infection) that received control plasmids

revealed dense infiltration of inflammatory cells, mainly of

macrophages, lymphocytes and epithelioid cells, and numerous

fungal cells (Fig. 5A). Around the foci of epithelioid granulomas,

giant cells were observed. In contrast, there were large areas of

normal lung architecture in pP10-immunized mice and a global

reduction in the number of granulomas with few yeast cells

(Fig. 5B). Treatment with pIL-12 resulted in histological findings

that were more similar to controls than to pP10-immunized mice

(Fig. 5C). Importantly, the lungs of mice treated with the

Figure 4. Long term therapeutic treatment of experimental PCM. Gene immunization started 30 days after infection and mice were sacrificed6 months after infection. CFUs were counted in lungs of B10.A mice infected intratracheally with 36105 yeast cells and immunized with vectorscontaining the insert encoding P10 (pP10) or IL-12 (pIL-12). Control mice were inoculated with PBS or with vector without insert. Each bar representsthe average counts and standard deviations of CFU in lungs from 5 to 10 animals in each group. Experiments were carried out in triplicate, withsimilar results. ** p#0.001, comparing vector with and without insert; ##p#0.001, comparing untreated and other groups.doi:10.1371/journal.pntd.0001519.g004



combination of pP10 and pIL-12 were mostly histologically

normal and no yeast cells were identified (Fig. 5D).

Cytokine assaysPrevious studies with BALB/c mice have established that P10

elicits a protective Th-1 immune response [11]. BALB/c and

B10.A mice have different genetic backgrounds that strongly

influence their response to infection by P. brasiliensis. Their

different susceptibility to fungal infection depends in part on their

capacity to produce pro-inflammatory cytokines, which are often

reduced in B10.A relative to BALB/c. IL-4, IL-10, IL-12 and IFN-

c were measured in the lungs of infected B10.A mice and BALB/c.

In mice subjected to the second protocol, BALB/c mice responded

to pcDNA3 and pP10 gene immunization with significant increase

in IFN-c in the lung homogenate compared to untreated or

pcDNA3 treated animals (data not shown). In contrast, B10.A

mice produced significantly less IFN-c after immunization with

pP10 and pcDNA3 in comparison to treatment with pcDNA3

gene alone, which suggests that the increase in IFN-c in both of

these groups relative to untreated mice could be due to dendritic

cell activation by plasmid CpGs. The cytokine production in the

group of animals submitted to the third protocol, in which B10.A

mice treated with pP10 with or without pIL-12 had undetectable

yeasts in the lung tissue, is shown in Table 1. After 6 months post-

infection, cytokine analyses in these mice showed a persistent IFN-

c production regulated by an IL-10-rich immune response, which

is compatible with a protective therapeutic effect in B10.A mice.

Discussion

A vaccine against P. brasiliensis using plasmid DNA was first

tested in 2000 [24,25]. BALB/c mice were immunized with a

Table 1. Cytokines in lung homogenates of B10.A miceinfected i.t. with P. brasiliensis Pb 18 yeasts and submitted togene immunization for 5 months.

Cytokines pg/ml IL-10 IFN-c

Untreated 12.1864.61 4.7161.75

pcDNA3 10.3866.53 4.7362.45

pP10+pcDNA3 18.7166.10 7.6462.46

pP10+pIL-12 12.0062.06 7.3161.37

Mice were sacrificed 1 month after the last dose.doi:10.1371/journal.pntd.0001519.t001

Figure 5. Histopathology of lungs from intratracheally infected B10.A mice. Animals were infected with P. brasiliensis for one month,treated with or without vectors carrying P10 or IL-12 DNA inserts according to protocol 3, and sacrificed 6 months after the initial infection. Infectedmice treated with (A) control pcDNA3, (B) pP10, (C) pIL-12 DNA, and (D) P10 and IL-12 DNA. Gomori staining; original magnification, 406.doi:10.1371/journal.pntd.0001519.g005



mammalian expression vector carrying the full gene of the gp43

under the control of CMV promoter with Freund’s adjuvant

resulting in the induction of both B and T cell-mediated immune

responses characterized by a mixed Th-1/Th-2 long-lasting

cellular immune response, chiefly modulated by IFN-c. This

immunization method was protective when performed in mice

prior to challenge with virulent P. brasiliensis. When tested for

immunoprotection, P10 in Freund’s adjuvant was also active in the

murine model of PCM, eliciting an IFN-c-dependent Th-1

immune response [11,20]. The combined treatment of P10-

vaccine in Freund’s adjuvant and chemotherapy, either an azole,

amphotericin B, or sulfamethoxazole, stimulated a protective Th-1

response, rich in IL-12 and IFN-c, that was therapeutically

beneficial if initiated 2 or 30 days after intratracheal infection [19].

The combined treatment was also effective in anergic animals

challenged with the fungus [20].

Presently we used a DNA vaccine encoding P10 with or without

a plasmid encoding IL-12 that is administered without adjuvant.

We found that the pP10 vaccine, either given prior to or 1 month

after intratracheal infection, induced a significant reduction in the

fungal burden in the lungs of mice. Co-vaccination with murine

pIL-12 significantly enhanced vaccine effectiveness, particularly in

a long- term infection model in B10.A mice. The combined DNA

vaccine (Protocol 3) achieved virtual sterilization after 6 months

with histologically normal lungs and undetectable fungal burden.

Full protection was mediated by IFN-c production and the pro-

inflammatory effect of pP10 and pIL-12 was regulated by IL-10 in

these susceptible mice.

The mechanism of fungal killing by gene immunization is not

solely mediated by cytokines since the empty plasmid pcDNA3 is a

strong stimulator of the immune system. However, a significant

protection is only achieved with pP10 or pP10+pIL-12 adminis-

tration. P10 is not protective in IFN-c-KO mice [12], indicating

that this cytokine is essential for fungal killing through macrophage

activation. T-CD4+ lymphocytes recognizing P10 and other cells

induced by fungal infection are the main producers of IFN-c. A

role for a simultaneous induction of protective antibodies against

fungal antigens [26] is also recognized.

IL-12 administration has been previously studied in experimental

PCM [27]. Our current results show that IL-12 protected mice

against disseminated infection. In the long term infection protocol,

pIL-12 alone was only partially effective in the protection of infected

mice, but the cytokine facilitated the elimination of P. brasiliensis

when combined with pP10. This is a very encouraging result and

strongly suggests that a pP10-based vaccine associated with pIL-12

could be used as a powerful adjuvant to chemotherapy.

Despite the effectiveness of chemotherapy, fatalities from

invasive or systemic fungal diseases are not uncommon. Vaccines

against fungal diseases are gaining increasing attention, owing to

their capacity to effectively modulate the immune response

(reviewed in [28]. The frequent occurrence of clinical relapses

and sequellae, such as pulmonary fibrosis, following antifungal

chemotherapy suggest that immunoprotective vaccines could also

reduce the incidence of these complications [29].

In addition to our work with P10, there have been other notable

attempts to develop vaccine strategies for the treatment of PCM.

They included a cDNA encoding the antigenic protein rPb27 [30],

the recombinant heat shock protein 60 emulsified in adjuvant [31],

radioattenuated P. brasiliensis yeast cells [32] and Mycobacterium

leprae DNAhsp65 plasmid in infected BALB/c mice [33]. Braga

et al. [34] immunized BALB/c mice either with recombinant

purified flagellins (FliC) genetically fused with P10 or with the

synthetic P10 peptide mixed with purified FliC. A prevailing Th1-

type immune response was obtained that reduced P. brasiliensis

growth and lung damage in infected mice.

From a practical standpoint, the broad use of antifungal

vaccines is not realistic when considering the perspective of a large

number of infected people relative to the number of individuals

who develop the disease. Mycoses caused by dimorphic fungi, such

as PCM, coccidioidomycosis, histoplasmosis and blastomycosis,

have low incidence as a deep-seated disease. Certain fungal

diseases such as cryptococcosis, aspergillosis and candidiasis

typically occur in immunocompromised hosts. Hence, targeted

prophylactic vaccination may be a more practical approach to

control disease. In the case of PCM, immunization of those at

highest risk, such as farmers in highly endemic regions would be

reasonable. However, we have also demonstrated that the pP10/

pIL-12 combination is highly efficacious after PCM has developed.

Therefore, immunization could be most useful in combination

with standard therapy in PCM patients in order to enhance

treatment efficacy, reduce treatment duration and, perhaps,

prevent relapses.

Author Contributions

Conceived and designed the experiments: CPT LRT. Performed the

experiments: GMGR JEM AFM. Analyzed the data: JDN CPT LRT.

Contributed reagents/materials/analysis tools: CPT. Wrote the paper:

CPT LRT JDN.

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MAYORGA, O.; MUÑOZ, J. E.; LINCOPAN, N.; TEIXEIRA, A,; FERREIRA, L. C. S.; TRAVASSOS, L.; TABORDA C. P. The role of adjuvants in therapeutic protection against paracoccidioidomycosis after immunization with the P10 peptide. Frontiers in Microbiology,

3:154. doi:10.3389/fmicb.2012.00154. 2012.

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ORIGINAL RESEARCH ARTICLEpublished: 04 May 2012

doi: 10.3389/fmicb.2012.00154

The role of adjuvants in therapeutic protection againstparacoccidioidomycosis after immunization with theP10 peptideOriana Mayorga1, Julian E. Muñoz1, Nilton Lincopan1,2, Aline F.Teixeira1, Luis C. S. Ferreira1,

Luiz R.Travassos3 and Carlos P.Taborda1,4*

1 Department of Microbiology, Biomedical Sciences Institute of University of São Paulo, São Paulo, São Paulo, Brazil2 Department of Clinical Analysis, School of Pharmacy, University of São Paulo, São Paulo, São Paulo, Brazil3 Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, São Paulo, São Paulo, Brazil4 Laboratory of Medical Mycology-LIM53/IMTSP, University of São Paulo, São Paulo, São Paulo, Brazil

Edited by:

Joshua D. Nosanchuk, Albert EinsteinCollege of Medicine, USA

Reviewed by:

Joshua D. Nosanchuk, Albert EinsteinCollege of Medicine, USALeonardo Nimrichter, FederalUniversity of Rio de Janeiro, Brazil

*Correspondence:

Carlos P. Taborda, Department ofMicrobiology, Biomedical SciencesInstitute of University of São Paulo,Av. Prof. Lineu Prestes, 1374, SãoPaulo, São Paulo 05008-900, Brazil.e-mail: [email protected]

Paracoccidioidomycosis (PCM), a common chronic mycosis in Latin America, is a gran-ulomatous systemic disease caused by the thermo-dimorphic fungus Paracoccidioidesbrasiliensis. The glycoprotein gp43 is the main antigen target of P. brasiliensis and a 15-mer internal peptide (QTLIAIHTLAIRYAN), known as P10, defines a major CD4+-specificT cell epitope. Previous results have indicated that, besides having a preventive rolein conventional immunizations prior to challenge with the fungus, protective anti-fungaleffects can be induced in P. brasiliensis-infected mice treated with P10 administered withcomplete Freund’s adjuvant (CFA). The peptide elicits an IFN-γ-dependent Th1 immuneresponse and is the main candidate for effective immunotherapy of patients with PCM,as an adjunctive approach to conventional chemotherapy. In the present study we testedthe therapeutic effects of P10 combined with different adjuvants [aluminum hydroxide,CFA, flagellin, and the cationic lipid dioctadecyl-dimethylammonium bromide (DODAB)] inBALB/c mice previously infected with the P. brasiliensis Pb18 strain. Significant reduc-tions in the number of colony forming units of the fungus were detected in lungs ofmice immunized with P10 associated with the different adjuvants 52 days after infec-tion. Mice treated with DODAB and P10, followed by mice treated with P10 and flagellin,showed the most prominent effects as demonstrated by the lowest numbers of viableyeast cells as well as reductions in granuloma formation and fibrosis. Concomitantly,secretion of IFN-γ and TNF-α, in contrast to interleukin (IL)-4 and IL-10, was enhancedin the lungs of mice immunized with P10 in combination with the tested adjuvants,with the best results observed in mice treated with P10 and DODAB. In conclusion,the present results demonstrate that the co-administration of the synthetic P10 pep-tide with several adjuvants, particularly DODAB, have significant therapeutic effects inexperimental PCM.

Keywords: Paracoccidioides brasiliensis, paracoccidioidomycosis, P10, adjuvants, dioctadecyl-dimethylammonium

bromide, FliC flagellin, aluminum hydroxide, complete Freund’s adjuvant

INTRODUCTIONParacoccidioidomycosis (PCM) is a systemic mycosis that typ-ically starts as a granulomatous pulmonary disease subsequentto the inhalation of conidia of the dimorphic fungus Paracoccid-ioides brasiliensis. When it is not diagnosed and treated properly,P. brasiliensis yeast cells can spread rapidly to lymph nodes, tegu-ment, spleen, liver, and lymphoid organs of the digestive tract(Shikanai-Yasuda et al., 2006). PCM is endemic in Latin America,mostly affecting rural workers in Brazil, Colombia, and Venezuela(Wanke and Londero, 1994), and the majorities are involved inagricultural activities (Blotta et al., 1999; Restrepo et al., 2008). InBrazil, approximately 1,853 (∼51.2%) of 3,583 confirmed deathsdue to systemic mycoses from 1996 to 2006 were caused by PCM(Prado et al., 2009).

gp43 is a glycoprotein of 416 amino acids (Puccia et al., 1986;Cisalpino et al., 1996). A specific T-CD4+ cell epitope was mappedto a 15-amino acid sequence designated P10, which is recognizedby T cells from mice infected with P. brasiliensis. Immunization ofpreviously intratracheally infected BALB/c mice with P10 reducesthe fungal load in the lungs more than 200-fold as comparedto non-immunized animals (Taborda et al., 1998). P10 immu-nized animals produced greater amounts of IFN-γ and interleukin(IL)-12. These mice also had significantly reduced damage tolung tissue. In fact, the immune response elicited by P10 pre-vents the rapid spread of P. brasiliensis, and we have hypothesizedthat P10 administered as with newer adjuvants might enhancethe immunoprotection by the peptide. Hence, we have begun toinvestigate the efficacy of different adjuvants co-administered with

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Mayorga et al. The role of adjuvants in therapeutic protection

P10. Along this line, we have found FliC flagellin, derived fromSalmonella enterica, can significantly modify the Th-1 immuneresponse associated with P10 (Braga et al., 2009).

Aluminum hydroxide (Alum) is another adjuvant widelyused in human and veterinary vaccines, which is highly effec-tive in eliciting primary immune responses to target antigens.Concerning secondary responses, the adjuvant stimulates a Th-2 immune response that mainly stimulates the production ofantibodies. In this sense it is perhaps less suitable for vac-cines against intracellular microorganisms (HogenEsch, 2002).Alum has been associated with severe local reactions such aserythema, subcutaneous nodules, and contact hypersensitivity(Baylor et al., 2002).

Promising results have been observed with complete Freund’sadjuvant (CFA) and P10 (Marques et al., 2008). However, thisadjuvant causes a variety of side effects such as localized injection-site granulomas, hepatic and renal granuloma formation, andnecrotizing dermatitis. Therefore, the use of CFA has been lim-ited to experimental immunizations in animal studies. Due tothe severity of adverse reactions, this adjuvant has been bannedfor use in humans as well as for non-experimental veterinaryadministration (Stills Jr., 2005).

The idea of using cationic lipids (dioctadecyl-dimethylammo-nium bromide, DODAB) as adjuvants arose from the effectiveuptake of microparticles by both dendritic cells and macrophages(Lincopan et al., 2009). Cationic polymer particles carry antigento these phagocytes and can efficiently stimulate antibody pro-duction and activate cytotoxic T cells at low antigen dose (Singhet al., 2000; Lincopan et al., 2009). DODAB also induces matu-ration of dendritic cells (Thiele et al., 2001; Little et al., 2004)with high levels of IL-12 and IFN-γ production, which may bean important benefit in the design of an anti-Paracoccidioidesvaccine.

In the present work, a comparative appraisal of the variousadjuvants is presented aiming to identify which compound pro-duces the most effective immune response to P10 using murinemodels of PCM.

MATERIALS AND METHODSANIMALSSix male BALB/c mice per group (6- to 8-week old) were housedin polypropylene cages under specific pathogen free conditions.Animals used in this study were bred at University of São Pauloanimal facility. All experiments involving animals were con-ducted and approved by the Ethics Committee of Universityof São Paulo and conducted in accordance with internationalrecommendations.

FUNGAL STRAINVirulent P. brasiliensis Pb18 yeast cells were used to infect theanimals. The strain was maintained by weekly passage on solidSabouraud medium at 37◦C and yeast cells were used after 7–10 days of growth. Before the experimental infection, the funguswas grown in modified McVeigh–Morton medium at 37◦C for 5–7 days (Restrepo and Arango, 1980). Fungal cells were washed inphosphate-buffered saline (PBS, pH 7.2) and counted in a hemo-cytometer. The viability of fungal suspensions was determined by

staining with trypan blue (Sigma, St. Louis, MO, USA) and wasalways higher than 90%. The virulence of the Pb18 strain waschecked in each experiment by infecting BALB/c mice i.t. andrecovering the yeast cells from the infected organs.

INTRATRACHEAL INFECTION OF BALB/c MICEBALB/c mice were inoculated i.t. with 3 × 105 virulent Pb18 yeastcells/animal, grown on Sabouraud agar and suspended in sterilesaline (0.85% NaCl). A maximum volume of 50 μl was inoculatedper mouse. Briefly, mice were anesthetized i.p. with 200 μl of asolution containing 80 mg/kg ketamine and 10 mg/kg of xylazine(both from União Química Farmacêutica, Brazil). After approx-imately 5 min, their necks were hyperextended, and the tracheaswere exposed at the level of the thyroid and injected with 3 × 105

yeast cells.

PEPTIDE SYNTHESIS AND PURIFICATIONPeptide synthesis and purification was carried out at the Depart-ment of Biophysics, UNIFESP as described previously (Tabordaet al., 1998). HPLC analysis showed that the synthetic P10 in theamidated form was >90% pure.

IMMUNIZATION OF MICEImmunization of BALB/c mice (6- to 8-week old males) was ini-tiated 30 days after infection and repeated on days 37 and 44, bythe subcutaneous route, with 20 μg of P10 in presence of therespective adjuvant. The adjuvants used were CFA with subse-quent immunizations with incomplete Freund’s adjuvant (IFA);Alum 100 μg/ml; FliC flagellin 5 μg/animal, and cationic lipidat 0.1 mM/animal. All adjuvants were vortexed with the peptidebefore immunization. The animals were sacrificed 7 days after thelast immunization, at day 52 of infection.

COLONY FORMING UNITSFor each mouse, the lungs, spleen, and liver were excised andweighed immediately after sacrifice. Tissues were individuallyhomogenized in PBS and 100 μl of this suspension was plated onbrain heart infusion medium (BHI; Difco Laboratories, Detroit,MI, USA), supplemented with 4% fetal bovine serum (Gibco,NY, USA) and 5% of the spent culture medium of P. brasilien-sis 192 isolate (Castañeda et al., 1988), streptomycin/penicillin10 IU/ml (Cultilab, Brazil), and cycloheximide 500 μg/ml (Sigma,St Louis, MO, USA). The plates were incubated at 37◦C for aperiod of 10 days. The numbers of colonies were counted andresults expressed in colony forming unit (CFU) per gram oftissue.

CYTOKINE ANALYSISLungs of each mouse were macerated with protease inhibitor(Sigma, St Louis, MO, USA) and centrifuged; supernatants ofthese samples were used for cytokine detection. IL-4, IL-10, TNF-α, and IFN-γ were measured using ELISA kits (BD Biosciences,San Diego, CA, USA). The detection limits of the assays were asfollows: 7.8 pg/ml for IL-4, 31.25 pg/ml for IL-10 and IFN-γ, and15.6 pg/ml for TNF-α, as previously determined by the manufac-turer. Cytokine levels present in the supernatant preparations wereanalyzed using GraphPad Prism 5.

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HISTOPATHOLOGYThe lungs were excised, fixed in 10% buffered formalin (Merck,Germany) and submitted to histopathological analysis [(hema-toxylin and eosin (H&E) and Masson’s trichrome].

STATISTICAL ANALYSISStatistics was done using GraphPad Prism 5 software (San Diego,CA, USA). The results were expressed as mean values and stan-dard deviations (SDs) of the indicated values. The non-parametricTukey’s honestly significant difference test was employed. p-Valuesof ≤0.05 indicated statistical significance.

RESULTSCOLONY FORMING UNITS IN INFECTED BALB/c MICE IMMUNIZEDWITH PEPTIDE 10 (P10)After 30 days of infection, immunizations were initiated with threeweekly doses of P10 with or without the different adjuvants. After52 days of infection, the fungal load was evaluated by enumerationof CFUs from recovered organs (lung, spleen, and liver). Lungsof mice immunized with P10 along with each of the differentadjuvants had a significantly reduced number of CFU comparedto controls (Figure 1). The order of efficacy for CFU reductionwas cationic lipid and P10 >> CFA/IFA > Alum > FliC. In thismodel, we did not recover any fungal cells from the spleens or liversof mice that received any of the immunizations with P10 (datanot shown), demonstrating the effectiveness of the peptide in thecontrol of experimental PCM. In contrast, the fungal burdens were625 ± 60 CFU/g of tissue in the spleens and 296 ± 59 CFU/g oftissue in the livers of unimmunized mice.

CYTOKINE PATTERN INDUCED BY IMMUNIZATION WITH P10ASSOCIATED WITH ADJUVANTSIn comparison with control infected animals, IFN-γ was signifi-cantly increased in mice that received P10 with either cationic lipidor FliC (Figure 2A). TNF-α levels were significantly increased onlyin mice treated with P10 and cationic lipid (Figure 2B). P10 immu-nization with either cationic lipid or FliC significantly reduced

FIGURE 1 | Colony forming units (CFU) from lungs of BALB/c mice

infected intratracheally with 3 × 105 yeast cells of Pb18 and

immunized at 30, 37, and 44 days after infection with the different

adjuvants with or without P10. Animals were sacrificed after 60 days ofinfection. Control animals were infected by not immunized (IFN). Theadjuvants used were: aluminum hydroxide alone (ALU) or with P10 (AP10),FliC flagellin alone (FLA) or with P10 (FP10), complete Freund’s adjuvantalone (CFA) or with P10 (CF10), and cationic lipid alone (CLI) or with P10(CP10). Significant difference *p < 0.05, **p < 0.01.

levels of IL-4 (Figure 2C) and IL-10 (Figure 2D). Immuniza-tion with P10 and Alum also reduced IL-10 levels, but did notsignificantly alter the levels of the other cytokines analyzed.

LUNG HISTOPATHOLOGY IN BALB/c MICE INFECTED (i.t.) WITH Pb18AND IMMUNIZED WITH P10Lung tissues from mice immunized with cationic lipid with orwithout P10 were stained with H&E and compared with unim-munized infected tissues as well as uninfected lungs (Figure 3).As expected, the non-infected group (Figure 3A) showed nor-mal lung tissue, and the unimmunized infected lungs (Figure 3B)showed dense cell infiltrates with high numbers of fungal cells dis-seminated throughout the lung parenchyma. In the case of miceimmunized only with the cationic lipid, we observed the forma-tion of loose granulomas with many fungal cells (Figure 3C).In contrast, lungs of mice immunized with cationic lipid andP10 showed significantly preserved lung parenchyma withoutfungal cells (Figure 3D). The histological appearances of lungsfrom mice immunized with either FliC (Figure 3E) or Alum(Figure 3G) alone were similar to that with cationic lipid alone(Figure 3C). Immunization of mice with P10 and FliC resulted inimproved granuloma formation in the lungs, which prevents thespread of the fungus, and increased preservation of normal lungparenchyma (Figure 3F). Although there was a slight increase innormal lung parenchyma in mice immunized with P10 and Alum,there was poor granuloma formation and large numbers of yeastcells within areas of inflammation (Figure 3H).

The amount of pulmonary collagen type I in mice immunizedwith P10 and FliC, Alum, or cationic lipid were compared withunimmunized infected controls using Masson’s trichrome stain-ing. Tissues from control mice revealed abundant collagen I fiberswithin cellular infiltrates containing large numbers of fungal cells(Figure 4A). Although no fungal cells were visualized, lungs ofmice immunized with FliC flagellin and P10 nevertheless diffuselydisplayed increased amounts of collagen (Figure 4B). Mice immu-nized with Alum and P10 showed large granulomas containingfungal cells and the formation of collagen fibers on the granu-loma’s periphery (Figure 4C). In contrast, mice immunized withcationic lipid and P10 displayed preserved lung tissue withoutincreased collagen (Figure 4D).

DISCUSSIONThe P10 peptide (QTLIAIHTLAIRYAN) has important immuno-protective properties that make it a leading candidate for thedevelopment of a therapeutic vaccine (Taborda et al., 1998). Wehave also shown that P10 immunization can be utilized concomi-tantly with standard antifungal drugs in the treatment of PCMand that co-administration may also prevent disease recurrence(Marques et al., 2008).

The protective effect of P10 is related to the induction of anINF-γ dependent Th-1 immune response (Taborda et al., 1998;Travassos et al., 2007), so it may be stimulated by adjuvants ornanoparticle encapsulation that increase the efficiency of P10uptake by dendritic cells resulting in the enhanced presentationof the peptide for cellular immune responses (Amaral et al., 2010;Magalhães et al., 2012). In the present work, we show that the asso-ciation of P10 with cationic lipids led to a significant reduction of




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FIGURE 2 | Cytokine detection was assayed in the lung tissue from

BALB/c mice 52 days after infection. Analyzed cytokines were: (A) IFN-γ,(B) TNF-α, (C) IL-4, and (D) IL-10. Each group was infected i.t. with 3 × 105

yeast cells and immunized at 30, 37, and 44 days after infection with differentadjutants with or without P10. The groups of mice included unimmunized,

infected control mice (INF); animals infected and immunized with aluminumhydroxide alone (ALU) or with P10 (AP10), FliC flagellin alone (FLA) or withP10 (FP10), and cationic lipid alone (CLI) or with P10 (CP10). *p < 0.05:significance p < 0.05 compared to control mice (only infected). **p < 0.01:compared to control mice (only infected).

CFU in the lungs of 52-day infected animals (Figure 1). In con-trast, animals immunized only with P10 (without adjuvants), hadminimal reductions in fungal burden (not shown data). Hence,combining adjuvants, such as cationic lipids, with P10 can gener-ate augmented immune responses mediated by Th-1 cells, directlyrelated to the secretion of IFN-γ (Figure 2A), which leads to peri-toneal and lung macrophage activation as well as enhancing theirfungicidal effect on yeasts and conidia of P. brasiliensis (Buissa-Filho et al., 2008). Immunization performed with P10 associatedwith antifungal drugs in animals infected with Pb18, has beenshown to induce a significant reduction in IL-4 (Marques et al.,2008), which we now show also occurs in the setting of immu-nizations with cationic lipid and P10 (Figure 2C). Moreover, P10administration with cationic lipid also significantly reduces levelsof IL-10 (Figure 2D). It is worth noting, however, that dependingon the degree of inflammation generated by the Th-1 response,Th-2 cytokines are essential for balancing the immune responseand reducing the risk of self-damage (Travassos et al., 2008).

Studies with cationic lipids associated with recombinant HSPof Mycobacterium leprae have demonstrated the ability of theadjuvant to promote antigen presentation in lymph node cellswith production of high levels of IL-12 and INF-γ, suggestingthat cationic lipid can be useful in the formulation of vaccinesagainst intracellular bacteria, as well as against protozoa (Linco-pan et al., 2009). IFN-γ is required for the synthesis of TNF-α bymacrophages and is essential for the accumulation of these cellsand their subsequent differentiation into epithelioid cells. INF-γis also responsible for granuloma’s formation and maintenance,and, therefore, it plays a vital role in the control of disseminationby fungi such as P. brasiliensis (Souto et al., 2000).

Although the cationic lipid/P10 association has demonstratedthe best protective response in the setting of short-term infectionin our murine model, it is relevant also to consider the protectiveeffect of the association of P10 and FliC flagellin. A significantreduction in the number of CFU was observed and the cytokineprofile was similar to that achieved with cationic lipid and P10




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FIGURE 3 | Histological sections of murine lungs from i.t. infected

BALB/c mice submitted to immunization with P10 associated to

cationic lipid. Groups of mice included (A) uninfected controls, (B)

unimmunized infected controls, (C) infected and immunized only with thecationic lipid, (D) infected and immunized with cationic lipid plus P10, (E)

infected and immunized only with FliC flagellin, (F) infected and immunizedwith FliC flagellin plus P10, (G) infected and immunized only with aluminumhydroxide, (H) infected and immunized with aluminum hydroxide plus P10after 52 days of infection. H&E staining, ×10 magnification.

(Figures 1 and 2). We have previously shown that prophylacticexperiments performed with FliC flagellin have demonstrated theeffectiveness of the association of this adjuvant with P10 allowingfor the control of fungal infection in vaccinated mice (Braga et al.,2009). Intranasal immunization carried out with this formula-tion induced high levels of IFN-γ and IL-12 production by lungcells and suppressed the production of Th-2 cytokines. The for-mation of compact granulomas (Figure 3F) as well as cytokinelevels obtained with FliC Flagellin and P10, can be associatedto the administration route, since present immunizations were

FIGURE 4 | Histological sections of murine lungs from i.t. infected

BALB/c mice immunized after 30 days of infection with P10 associated

with FliC flagellin, aluminum hydroxide, or cationic lipid. (A) infected,unimmunized control mice, (B) infected and immunized with FliC flagellinplus P10, (C) infected and immunized with aluminum hydroxide plus P10,and (D) infected and immunized with cationic lipid plus P10 after 52 days ofinfection. Masson’s trichrome staining, ×40 magnification. Blue stainingand arrows indicate type I collagen fibers.

performed subcutaneously, according to Braga et al. (2009), betterresults are obtained with intranasal immunizations.

In the lung parenchyma, P. brasiliensis induces chronic dam-age leading to the development of pulmonary fibrosis, which ispresumably due to persistent antigenic stimulation and an ongo-ing active immune response. Granulomatous inflammation canincrease the formation of connective tissue rich in collagen typeI and III, leading to functional changes and subsequent fibrosisin the lung (Naranjo et al., 2010). Since fibrosis is a well-knownsequela of PCM, it is therefore important that the adjuvants useddo not induce an exacerbated inflammatory response. In fact,the association of cationic lipid and P10 resulted in a signifi-cant reduction of pulmonary fibrosis in mice infected with Pb18(Figure 4D).

In summary, we have shown that the examined regimens com-bining P10 with different adjuvants results in significantly differentprotective responses. Administration of P10 with cationic lipidprovided the most effective response profile, with the greatestreduction in fungal burden and a Th-1 biased cytokine responsethat maintained pulmonary architecture without inducing fibroticinjury.

ACKNOWLEDGMENTSThis work was supported by grants from FAPESP 11/17267-4,09/15823-7, 09/53354-9, 07/58750-4 and CNPq 146809/2010-6.Carlos P. Taborda, Luiz R. Travassos, Luis C. S. Ferreira, and NiltonLincopan are research fellows of the CNPq. We acknowledge thevaluable scientific inputs from Dr. Joshua D. Nosanchuk.




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Conflict of Interest Statement: Theauthors declare that the research wasconducted in the absence of any com-mercial or financial relationships thatcould be construed as a potential con-flict of interest.

Received: 02 April 2012; accepted: 03April 2012; published online: 04 May2012.Citation: Mayorga O, Muñoz JE, Lin-copan N, Teixeira AF, Ferreira LCS,Travassos LR and Taborda CP (2012)The role of adjuvants in therapeuticprotection against paracoccidioidomyco-sis after immunization with the P10peptide. Front. Microbio. 3:154. doi:10.3389/fmicb.2012.00154This article was submitted to Frontiers inFungi and Their Interactions, a specialtyof Frontiers in Microbiology.Copyright © 2012 Mayorga, Muñoz,Lincopan, Teixeira, Ferreira, Travas-sos and Taborda. This is an open-access article distributed under the termsof the Creative Commons AttributionNon Commercial License, which per-mits non-commercial use, distribution,and reproduction in other forums, pro-vided the original authors and source arecredited.


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